U.S. patent application number 12/997471 was filed with the patent office on 2011-09-08 for p2/p2a/p2b gene sequences as diagnostic targets for the identification of fungal and yeast species.
This patent application is currently assigned to National University of Ireland Galway. Invention is credited to Thomas Gerard Barry, Marcin Jankiewicz, Sinead Lahiff, Majella Maher, Louise O'Connor, Terry James Smith, Nina Tuite.
Application Number | 20110217703 12/997471 |
Document ID | / |
Family ID | 41268781 |
Filed Date | 2011-09-08 |
United States Patent
Application |
20110217703 |
Kind Code |
A1 |
Barry; Thomas Gerard ; et
al. |
September 8, 2011 |
P2/P2A/P2B GENE SEQUENCES AS DIAGNOSTIC TARGETS FOR THE
IDENTIFICATION OF FUNGAL AND YEAST SPECIES
Abstract
The present invention relates to nucleic acid primers and probes
to detect one or more fungal and yeast species. More specifically
the invention relates to the P2, P2A and P2B gene sequences (also
known as 60S acidic ribosomal protein P2, RLA-2-ASPFU, Allergen ASP
f8 or Afp2), the corresponding RNA, specific probes, primers and
oligonucleotides related thereto and their use in diagnostic assays
to detect and/or discriminate fungal and yeast species.
Inventors: |
Barry; Thomas Gerard;
(Kinarva, IE) ; Smith; Terry James; (Galway,
IE) ; Jankiewicz; Marcin; (Galway, IE) ;
O'Connor; Louise; (Galway, IE) ; Tuite; Nina;
(Galway, IE) ; Lahiff; Sinead; (Gort, IE) ;
Maher; Majella; (Moycullen, IE) |
Assignee: |
National University of Ireland
Galway
|
Family ID: |
41268781 |
Appl. No.: |
12/997471 |
Filed: |
June 15, 2009 |
PCT Filed: |
June 15, 2009 |
PCT NO: |
PCT/EP2009/057338 |
371 Date: |
May 9, 2011 |
Current U.S.
Class: |
435/6.11 ;
536/24.32 |
Current CPC
Class: |
C12Q 1/6895
20130101 |
Class at
Publication: |
435/6.11 ;
536/24.32 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 13, 2008 |
IE |
2008/0489 |
Claims
1. A diagnostic kit for a yeast or fungal species comprising an
oligonucleotide probe capable of binding to at least a portion of
the P2, P2A or P2B genes or its corresponding mRNA.
2. A kit as claimed in claim 1 wherein the portion of the P2 gene
is selected from: a portion of the region of the gene from base
pair position 1 to base pair position 326 of the Aspergillus P2B
gene, a portion of base pair position 1 to base pair position 359
of the C. albicans P2B gene, a portion of base pair position 114 to
base pair position 439 of the C. albicans P2A gene, or a portion of
the region of the gene from base pair 24 to base pair position 158
in C. glabrata.
3. The kit of claim 1, comprising a probe for one or more of a
portion of the region of the gene from base pair position 1 to base
pair position 326 of the Aspergillus P2 gene, a portion of the
region of the gene from base pair 24 to base pair position 158 in
C. glabrata, a probe for a portion of base pair position 1 to base
pair position 359 of the C. albicans P2B gene and a probe for a
portion of base pair position 114 to by position 439 of the C.
albicans P2A gene.
4. The kit of claim 1, wherein the probe is selected from SEQ ID NO
9, 10, 91-94, 105-110, or sequences substantially similar or
complementary thereto which can also act as a probe.
5. The kit of claim 1, further comprising a primer for
amplification of at least a portion of the P2, P2A and/or P2B
gene.
6. The kit of claim 1, comprising a forward and a reverse primer
for a portion of the P2, P2A and/or P2B gene.
7. The kit of claim 1, comprising at least one forward in vitro
amplification primer and at least one reverse in vitro
amplification primer, the forward amplification primer being
selected from the group consisting of SEQ ID NO 1, 3, 7, 85-88,
95-99, or sequences substantially similar or complementary thereto
which can also act as a forward amplification primer and the
reverse amplification primer being selected from the group
consisting of SEQ ID NO 2, 4, 5, 6, 8, 89, 90, 100-104 or sequences
substantially similar or complementary thereto which can also act
as a reverse amplification primer.
8. The kit of claim 1, based on direct nucleic acid detection
technologies, signal amplification nucleic acid detection
technologies, and nucleic acid in vitro amplification technologies
is selected from one or more of Polymerase Chain Reaction (PCR),
Ligase Chain Reaction (LCR), Nucleic Acids Sequence Based
Amplification (NASBA), Strand Displacement Amplification (SDA),
Transcription Mediated Amplification (TMA), Branched DNA technology
(bDNA) and Rolling Circle Amplification Technology (RCAT) or other
enzymatic in vitro amplification based technologies.
9. A nucleic acid molecule selected from the group consisting of:
SEQ ID NO 1 through SEQ ID NO 110 and sequences substantially
homologous or substantially complementary thereto or to a portion
thereof and having a function in diagnostics based on the P2, P2A
and/or P2B genes.
10. A nucleic acid molecule comprising an oligonucleotide having a
sequence substantially homologous to or substantially complementary
to a portion of a nucleic acid molecule as claimed in claim 9.
11. A method of detecting a target organism in a test sample
comprising the steps of: (i) Mixing the test sample with at least
one oligonucleotide probe capable of binding to at least a portion
of the P2, P2A and/or P2B gene or its corresponding mRNA under
appropriate conditions; (ii) hybridizing under a high stringency
conditions any nucleic acid that may be present in the test sample
with the oligonucleotide to form a probe:target duplex; and (iii)
determining whether a probe:target duplex is present; the presence
of the duplex positively identifying the presence of the target
organism in the test sample.
15. A method as claimed in claim 11 wherein the probe is selected
from the group consisting of SEQ ID NO 9, 10, 91-94, 105-110 or
sequences substantially homologous or substantially complementary
thereto also capable of acting as a probe for the P2B gene.
16. The method of claim 11, wherein the target organism is a yeast
and/or fungal species.
17. Use of a kit of claim 1 in a diagnostic assay to measure yeast
and/or fungal titres in a patient.
18. A method of assessing the efficacy of a treatment regime
designed to reduce yeast and/or fungal titre in a patient
comprising use of a kit of claim 1, at one or more key stages of
the treatment regime.
19. Use of a kit of claim 1, in a diagnostic assay to measure yeast
and or fungal contamination in an environment.
20. Use as claimed in claim 19, wherein the environment is a
hospital, a food sample, an environmental sample e.g. water, an
industrial sample such as an in-process sample or an end product
requiring bioburden or quality assessment.
21. Use of a kit of claim 1, in the identification and/or
characterization of one or more disruptive agents that can be used
to disrupt the P2, P2A or P2B gene function.
22. Use as claimed in claim 21, wherein the disruptive agent is
selected from the group consisting of antisense RNA, PNA,
siRNA.
23-25. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to nucleic acid primers and
probes to detect one or more fungal and yeast species. More
specifically the invention relates to the P2, P2A and P2B gene
sequences (also known as 60S acidic ribosomal protein P2,
RLA-2-ASPFU, Allergen ASP f8 or Afp2), the corresponding RNA,
specific probes, primers and oligonucleotides related thereto and
their use in diagnostic assays to detect and/or discriminate fungal
and yeast species.
BACKGROUND TO THE INVENTION
[0002] Yeast and fungal infections represent a major cause of
morbidity and mortality among immunocompromised patients. The
number of immunocompromised patients at risk of yeast and fungal
infection continues to increase each year, as does the spectrum of
fungal and yeast agents causing disease. Mortality from fungal
infections, particularly invasive fungal infections, is 30% or
greater in certain risk groups. The array of available anti-fungal
agents is growing; however, so too is the recognition of both
intrinsic and emerging resistance to antifungal drugs. These
factors are contributing to the increased need for cost containment
in laboratory testing and have led to laboratory consolidation in
testing procedures.
[0003] Invasive fungal infections are on the increase. In 2003, it
was estimated that there were 9 million at risk patients of which
1.2 million developed infection. Candida spp. and Aspergillus spp.
now rank as the most prominent pathogens infecting immunosupressed
patients. In particular, infections are common in the urinary
tract, the respiratory system and the bloodstream, at the site of
insertion of stents, catheters and orthopaedic joints.
Approximately, 10% of the known Candida spp. have been implicated
in human infection. Invasive candidiasis occurs when candida enters
the bloodstream and it is estimated to occur at a frequency of
8/100,000 population in the US with a mortality rate of 40%.
Candida albicans is the 4.sup.th most common cause of bloodstream
infection. Aspergillosis usually begins as a pulmonary infection
that can progress to a life-threatening invasive infection in some
patients and has a mortality rate of greater than 90%. Emerging
mycoses agents include Fusarium, Scedosporium, Zygomycetes and
Trichosporon spp. ("Stakeholder Insight: Invasive fungal
infections", Datamonitor, January 2004).
[0004] Immunocompromised patients including transplant and surgical
patients, neonates, cancer patients, diabetics and those with
HIV/AIDs are at high risk of developing invasive fungal infections
(Datamonitor report: Stakeholder opinion--Invasive fungal
infections, options outweigh replacements 2004). A large number of
severe cases of sepsis are reported each year. Despite improvements
in its medical management, sepsis still constitutes one of the
greatest challenges in intensive care medicine. Microorganisms
(bacteria, fungi and yeast) responsible for causing sepsis are
traditionally detected in hospital laboratories with the aid of
microbiological culture methods with poor sensitivity (25-82%),
which are very time-consuming, generally taking from two to five
days to complete, and up to eight days for the diagnosis of fungal
infections. Definitive diagnosis of an infection caused by a yeast
or fungus is usually based on either, the recovery and
identification of a specific agent from clinical specimens or
microscopic demonstration of fungi with distinct morphological
features. However, there are numerous cases where these methods
fail to provide conclusive proof as to the infecting agent. In
these instances, the detection of specific host antibody responses
can be used, although again this can be affected by the immune
status of the patient. Time is critical in the detection and
identification of bloodstream infections typically caused by
bacteria and fungi. Effective treatment depends on finding the
source of infection and making appropriate decisions about
antibiotics or antifungals quickly and efficiently. Only after
pathogens are correctly identified can targeted therapy using a
specific antibiotic or anti-fungal begin. Many physicians would
like to see the development of better in vitro amplification and
direct detection diagnostic techniques for the early diagnosis of
yeast and fungi ("Stakeholder Insight: Invasive fungal infections",
Datamonitor, January 2004). Recently Roche.TM. launched a real time
PCR based assay (Septifast.TM.), for the detection of bacterial,
fungal and yeast DNA in clinical samples. Therefore, there is a
clear need for the development of novel rapid diagnostic tests for
clinically significant bacterial and fungal pathogens for
bioanalysis applications in the clinical sector. This has led the
current inventors to identify novel fungal and yeast nucleic acid
targets for application in Nucleic Acid Diagnostics (NAD) tests.
Fungal and yeast nucleic acid based diagnostics have focused
heavily on the ribosomal RNA (rRNA) genes, RNA transcripts, and
their associated DNA/RNA regions. The rRNA genes are highly
conserved in all fungal species and they also contain divergent and
distinctive intergenic transcribed spacer regions. Ribosomal rRNA
comprises three genes: the large sub-unit gene (28S), the small
sub-unit gene (18S) and the 5.8S gene. The 28S and 18S rRNA genes
are separated by the 5.8S rRNA and two internal transcribed spacers
(ITS 1 and ITS2). Because the ITS region contains a high number of
sequence polymorphisms, numerous researchers have concentrated
their efforts on these as targets (Atkins and Clark, 2004). rRNA
genes are also multicopy genes with >10 copies within the fungal
genome.
[0005] A number of groups are working on developing new assays for
fungal and yeast infections. U.S. 2004044193 relates to, amongst a
number of other aspects, the transcription factor CaTEC1 of Candida
albicans; inhibitors thereof, and methods for the diagnosis and
therapy of diseases which are connected with a Candida infection;
and also diagnostic and pharmaceutical compositions which contain
the nucleotide sequences, proteins, host cells and/or antibodies.
WO0183824 relates to hybridization assay probes and accessory
oligonucleotides for detecting ribosomal nucleic acids from Candida
albicans and/or Candida dubliniensis. U.S. Pat. No. 6,017,699 and
U.S. Pat. No. 5,426,026 relate to a set of DNA primers, which can
be used to amplify and speciate DNA from five medically important
Candida species. U.S. Pat. No. 6,747,137 discloses sequences useful
for diagnosis of Candida infections. EP 0422872 and U.S. Pat. No.
5,658,726 disclose probes based on 18S rRNA genes, and U.S. Pat.
No. 5,958,693 discloses probes based on 28S rRNA, for diagnosis of
a range of yeast and fungal species. U.S. Pat. No. 6,017,366
describes sequences based on chitin synthase gene for use in
nucleic acid based diagnostics for a range of Candida species. It
is clear though, that development of faster, more accurate
diagnostic methods are required, particularly in light of the
selection pressure caused by modern anti-microbial treatments which
give rise to increased populations of resistant virulent strains
with mutated genome sequences. Methods that enable early diagnosis
of microbial causes of infection enable the selection of a specific
narrow spectrum antibiotic or antifungal to treat the infection
(Datamonitor report: Stakeholder opinion--Invasive fungal
infections, options outweigh replacements 2004; Datamonitor report:
Stakeholder Opinion--Sepsis, under reaction to an overreaction,
2006).
[0006] Ribosomes are a two part organelle, composed of a large and
a small subunit. The large subunit possesses a number of features
which are conserved in all organisms for example the presence of a
complex of a number of acidic proteins, the P proteins which form a
stalk-like structure (Abramczyk et al., 2004 Tchorzewski et al.,
2000; 2003). In the 60S ribosomal subunit of eukaryotes these
acidic P proteins include, P0, P1 and P2 (Wool et al 1991). The
complex formed by these P proteins is required for efficient
translation. In yeasts, there are two isoforms of P1 (P1A and P1B)
and P2 (P2A and P2B) which are encoded by four distinct single copy
genes (Newton et al, 1990, Bailey-Serres et al., 1997).
[0007] This invention relates to the use of the P2A, P2B genes in
yeast and the P2 gene in fungi. There are currently 65 P2B gene
sequences (approx. 360 bp) available in NCBI GenBank database
including 3 P2B sequences for Candida albicans and one sequence of
high homology for C. glabrata. There are 329 P2 sequences available
in NCBI GenBank database including annotated sequences for two
Aspergillus species and presumptive P2 sequences for three species.
Additional P2B sequences of representative species of Candida and
P2 sequences of representative species of Aspergillus were
generated by the inventors following the design of PCR primers to
amplify P2B gene regions in Candida and P2 gene regions in
Aspergillus spp. The published and newly generated P2B and P2 gene
sequences for Candida and Aspergillus spp. respectively, were
aligned and analysed using bioinformatics tools. PCR primers and
species-specific DNA probes for selected species, Candida glabrata
and Aspergillus fumigatus were designed and demonstrated for
molecular species identification using real-time PCR. These
examples demonstrate the potential of P2A/P2B and P2 genes for
molecular identification of species of Candida, Aspergillus and
other yeast and fungal species.
Definitions
[0008] "Synthetic oligonucleotide" refers to molecules of nucleic
acid polymers of 2 or more nucleotide bases that are not derived
directly from genomic DNA or live organisms. The term synthetic
oligonucleotide is intended to encompass DNA, RNA, and DNA/RNA
hybrid molecules that have been manufactured chemically, or
synthesized enzymatically in vitro.
[0009] An "oligonucleotide" is a nucleotide polymer having two or
more nucleotide subunits covalently joined together.
Oligonucleotides are generally about 10 to about 100 nucleotides.
The sugar groups of the nucleotide subunits may be ribose,
deoxyribose, or modified derivatives thereof such as OMe. The
nucleotide subunits may be joined by linkages such as
phosphodiester linkages, modified linkages or by non-nucleotide
moieties that do not prevent hybridization of the oligonucleotide
to its complementary target nucleotide sequence. Modified linkages
include those in which a standard phosphodiester linkage is
replaced with a different linkage, such as a phosphorothioate
linkage, a methylphosphonate linkage, or a neutral peptide linkage.
Nitrogenous base analogs also may be components of oligonucleotides
in accordance with the invention. A "target nucleic acid" is a
nucleic acid comprising a target nucleic acid sequence. A "target
nucleic acid sequence," "target nucleotide sequence" or "target
sequence" is a specific deoxyribonucleotide or ribonucleotide
sequence that can be hybridized to a complementary
oligonucleotide.
[0010] An "oligonucleotide probe" is an oligonucleotide having a
nucleotide sequence sufficiently complementary to its target
nucleic acid sequence to be able to form a detectable hybrid
probe:target duplex under high stringency hybridization conditions.
An oligonucleotide probe is an isolated chemical species and may
include additional nucleotides outside of the targeted region as
long as such nucleotides do not prevent hybridization under high
stringency hybridization conditions. Non-complementary sequences,
such as promoter sequences, restriction endonuclease recognition
sites, or sequences that confer a desired secondary or tertiary
structure such as a catalytic active site can be used to facilitate
detection using the invented probes. An oligonucleotide probe
optionally may be labelled with a detectable moiety such as a
radioisotope, a fluorescent moiety, a chemiluminescent, a
nanoparticle moiety, an enzyme or a ligand, which can be used to
detect or confirm probe hybridization to its target sequence.
Oligonucleotide probes are preferred to be in the size range of
from about 10 to about 100 nucleotides in length, although it is
possible for probes to be as much as and above about 500
nucleotides in length, or below 10 nucleotides in length.
[0011] A "hybrid" or a "duplex" is a complex formed between two
single-stranded nucleic acid sequences by Watson-Crick base
pairings or non-canonical base pairings between the complementary
bases. "Hybridization" is the process by which two complementary
strands of nucleic acid combine to form a double-stranded structure
("hybrid" or "duplex"). A "fungus" or "yeast" is meant any organism
of the kingdom Fungi, and preferably, is directed towards any
organism of the phylum Ascomycota. "Complementarity" is a property
conferred by the base sequence of a single strand of DNA or RNA
which may form a hybrid or double-stranded DNA:DNA, RNA:RNA or
DNA:RNA through hydrogen bonding between Watson-Crick base pairs on
the respective strands. Adenine (A) ordinarily complements thymine
(T) or uracil (U), while guanine (G) ordinarily complements
cytosine (C).
[0012] The term "stringency" is used to describe the temperature,
ionic strength and solvent composition existing during
hybridization and the subsequent processing steps. Those skilled in
the art will recognize that "stringency" conditions may be altered
by varying those parameters either individually or together. Under
high stringency conditions only highly complementary nucleic acid
hybrids will form; hybrids without a sufficient degree of
complementarity will not form. Accordingly, the stringency of the
assay conditions determines the amount of complementarity needed
between two nucleic acid strands forming a hybrid. Stringency
conditions are chosen to maximize the difference in stability
between the hybrid formed with the target and the non-target
nucleic acid. With "high stringency" conditions, nucleic acid base
pairing will occur only between nucleic acid fragments that have a
high frequency of complementary base sequences (for example,
hybridization under "high stringency" conditions, may occur between
homologs with about 85-100% identity, preferably about 70-100%
identity). With medium stringency conditions, nucleic acid base
pairing will occur between nucleic acids with an intermediate
frequency of complementary base sequences (for example,
hybridization under "medium stringency" conditions may occur
between homologs with about 50-70% identity). Thus, conditions of
"weak" or "low" stringency are often required with nucleic acids
that are derived from organisms that are genetically diverse, as
the frequency of complementary sequences is usually less.
[0013] `High stringency` conditions are those equivalent to binding
or hybridization at 42.degree. C. in a solution consisting of
5.times.SSPE (43.8g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4H.sub.2O and
1.85 g/l EDTA, ph adjusted to 7.4 with NaOH), 0.5% SDS, 5.times.
Denhardt's reagent and 100 .mu.g/l denatured salmon sperm DNA
followed by washing in a solution comprising 0.1.times.SSPE, 1.0%
SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is used.
[0014] `Medium stringency` conditions are those equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times.Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 1.0.times.SSPE,
1.0% SDS at 42.degree. C., when a probe of about 500 nucleotides in
length is used.
[0015] `Low stringency` conditions are those equivalent to binding
or hybridization at 42.degree. C. in a solution consisting of
5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4H.sub.2O and
1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS,
5.times.Denhardt's reagent [50.times. Denhardt's contains per 500
ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)]
and 100 .mu.g/ml denatured salmon sperm DNA followed by washing in
a solution comprising 5.times.SSPE, 0.1% SDS at 42.degree. C., when
a probe of about 500 nucleotides in length is used.
[0016] In the context of nucleic acid in-vitro amplification based
technologies, "stringency" is achieved by applying temperature
conditions and ionic buffer conditions that are particular to that
in-vitro amplification technology. For example, in the context of
PCR and real-time PCR, "stringency" is achieved by applying
specific temperatures and ionic buffer strength for hybridisation
of the oligonucleotide primers and, with regards to real-time PCR
hybridisation of the probe/s, to the target nucleic acid for
in-vitro amplification of the target nucleic acid.
[0017] One skilled in the art will understand that substantially
corresponding probes of the invention can vary from the referred-to
sequence and still hybridize to the same target nucleic acid
sequence. This variation from the nucleic acid may be stated in
terms of a percentage of identical bases within the sequence or the
percentage of perfectly complementary bases between the probe and
its target sequence. Probes of the present invention substantially
correspond to a nucleic acid sequence if these percentages are from
about 100% to about 80% or from 0 base mismatches in about 10
nucleotide target sequence to about 2 bases mismatched in an about
10 nucleotide target sequence. In preferred embodiments, the
percentage is from about 100% to about 85%. In more preferred
embodiments, this percentage is from about 90% to about 100%; in
other preferred embodiments, this percentage is from about 95% to
about 100%
[0018] By "sufficiently complementary" or "substantially
complementary" is meant nucleic acids having a sufficient amount of
contiguous complementary nucleotides to form, under high stringency
hybridization conditions, a hybrid that is stable for detection.
The terms "identical" or percent "identity," in the context of two
or more nucleic acids or polypeptide sequences, refer to two or
more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or higher identity over a specified region, when compared and
aligned for maximum correspondence over a comparison window or
designated region) as measured using a BLAST or BLAST 2.0 sequence
comparison algorithms with default parameters described below, or
by manual alignment and visual inspection (see, e.g., NCBI web site
at ncbi.nlm.nih.gov/BLAST/or the like). Such sequences are then
said to be "substantially identical." This definition also refers
to, or may be applied to, the compliment of a test sequence. The
definition also includes sequences that have deletions and/or
additions, as well as those that have substitutions. As described
below, the preferred algorithms can account for gaps and the like.
Preferably, identity exists over a region that is at least about 25
amino acids or nucleotides in length, or more preferably over a
region that is 50-100 amino acids or nucleotides in length.
[0019] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Preferably, default program parameters can be used,
or alternative parameters can be designated. The sequence
comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference
sequence, based on the program parameters.
[0020] A "comparison window," as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Current Protocols in
Molecular Biology (Ausubel et al., eds. 1987-2005, Wiley
Interscience)). A preferred example of algorithm that is suitable
for determining percent sequence identity and sequence similarity
are the BLAST and BLAST 2.0 algorithms, which are described in
Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul
et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and
BLAST 2.0 are used, with the parameters described herein, to
determine percent sequence identity for the nucleic acids and
proteins of the invention. Software for performing BLAST analyses
is publicly available through the National Center for Biotechnology
Information. This algorithm involves first identifying high scoring
sequence pairs (HSPs) by identifying short words of length W in the
query sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, M=5, N=-4 and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a wordlength of
3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915
(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and
a comparison of both strands.
[0021] "Nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form, and complements thereof. The term encompasses
nucleic acids containing known nucleotide analogs or modified
backbone residues or linkages, which are synthetic, naturally
occurring, and non-naturally occurring, which have similar binding
properties as the reference nucleic acid, and which are metabolized
in a manner similar to the reference nucleotides. Examples of such
analogs include, without limitation, phosphorothioates,
phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,
2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
[0022] By "nucleic acid hybrid" or "probe:target duplex" is meant a
structure that is a double-stranded, hydrogen-bonded structure,
preferably about 10 to about 100 nucleotides in length, more
preferably 14 to 50 nucleotides in length, although this will
depend to an extent on the overall length of the oligonucleotide
probe. The structure is sufficiently stable to be detected by means
such as chemiluminescent or fluorescent light detection,
autoradiography, electrochemical analysis or gel electrophoresis.
Such hybrids include RNA:RNA, RNA:DNA, or DNA:DNA duplex
molecules.
[0023] "RNA and DNA equivalents" refer to RNA and DNA molecules
having the same complementary base pair hybridization properties.
RNA and DNA equivalents have different sugar groups (i.e., ribose
versus deoxyribose), and may differ by the presence of uracil in
RNA and thymine in DNA. The difference between RNA and DNA
equivalents do not contribute to differences in substantially
corresponding nucleic acid sequences because the equivalents have
the same degree of complementarity to a particular sequence.
[0024] By "preferentially hybridize" is meant that under high
stringency hybridization conditions oligonucleotide probes can
hybridize their target nucleic acids to form stable probe:target
hybrids (thereby indicating the presence of the target nucleic
acids) without forming stable probe:non-target hybrids (that would
indicate the presence of non-target nucleic acids from other
organisms). Thus, the probe hybridizes to target nucleic acid to a
sufficiently greater extent than to non-target nucleic acid to
enable one skilled in the art to accurately detect the presence of
(for example Candida) and distinguish these species from other
organisms. Preferential hybridization can be measured using
techniques known in the art and described herein.
[0025] By "theranostics" is meant the use of diagnostic testing to
diagnose the disease, choose the correct treatment regime and
monitor the patient response to therapy. The theranostics of the
invention may be based on the use of an NAD assay of this invention
on samples, swabs or specimens collected from the patient.
OBJECT OF THE INVENTION
[0026] It is an object of the current invention to provide
sequences and/or diagnostic assays to detect and identify one or
more yeast and fungal species. The current inventors have made use
of the P2, P2A and P2B gene sequences to design primers that are
specific to Candida P2B genes and to Aspergillus P2 genes. Such
primers not only allow the detection of yeast and fungal species
but also allow distinction between Candida and Aspergillus spp.
This has an advantage over the prior art in that if one wants to
identify a fungal pathogen in a sample, which contains Candida as a
commensal, the approach of using universal primers may not be
successful. There is a strong possibility that the Candida will
out-compete the fungal pathogen in the amplification process and
will be preferentially amplified, resulting in failure to detect
the disease-causing pathogen. The current invention further
provides for primers and probes that allow discrimination between
different Candida species and among different Aspergillus
species.
SUMMARY OF THE INVENTION
[0027] The present invention provides for a diagnostic kit for
detection and identification of yeast and/or fungal species,
comprising an oligonucleotide probe capable of binding to at least
a portion of the P2, P2A or P2B gene or its corresponding mRNA. The
oligonucleotide probe may have a sequence substantially homologous
to or substantially complementary to a portion of the P2, P2A or
P2B gene or its corresponding mRNA. It will thus be capable of
binding or hybridizing with a complementary DNA or RNA molecule.
The P2 gene may be a fungal P2 gene. The P2A gene may be a yeast
gene. The P2B gene may be yeast P2B gene. The nucleic acid molecule
may be synthetic. The oligonucleotide probe may have a sequence of
SEQ ID NO 9, 10, 91-94, 105-109, or a sequence substantially
homologous to or substantially complementary to those sequences,
which can also act as a probe for the P2, P2A or P2B genes.
[0028] The kit may comprise more than one such probe. In particular
the kit may comprise a plurality of such probes. In addition, the
kit may comprise additional probes for other organisms, such as,
for example, bacterial species or viruses.
[0029] The identified sequences are suitable not only for in vitro
DNA/RNA amplification based detection systems but also for signal
amplification based detection systems. Furthermore, the sequences
of the invention identified as suitable targets provide the
advantages of having significant intragenic sequence heterogeneity
in some regions, which is advantageous and enables aspects of the
invention to be directed towards group or species-specific targets,
and also having significant sequence homogeneity in some regions,
which enables aspects of the invention to be directed towards
genus-specific yeast and fungal primers and probes for use in
direct nucleic acid detection technologies, signal amplification
nucleic acid detection technologies, and nucleic acid in vitro
amplification technologies for yeast and fungal diagnostics. The
P2, P2A and P2B sequences allow for multi-test capability and
automation in diagnostic assays. One of the advantages of the
sequences of the present invention is that the intragenic P2, P2A
and P2B nucleotide sequences diversity between closely related
yeast or fungal species enables specific primers and probes for use
in diagnostics assays for the detection of yeast and fungi to be
designed. The P2, P2A and P2B nucleotide sequences, both DNA and
RNA can be used with direct detection, signal amplification
detection and in vitro amplification technologies in diagnostics
assays. The P2, P2A and P2B sequences allow for multi-test
capability and automation in diagnostic assays.
[0030] The kit may further comprise a primer for amplification of
at least a portion of the P2 and/or P2A and/or P2B genes. Suitably,
the kit comprises a forward and a reverse primer for a portion of
the P2, P2A and/or P2B gene.
[0031] The portion of the P2A gene may be equivalent to a portion
of the region of the gene from by position 114 to by position 439
of C. albicans (AF317661.1).
[0032] The portion of the P2B gene may be equivalent to a portion
of the region of the gene from base pair position 1 to position 335
in C. albicans (XM.sub.--718047.1, XM.sub.--717900.1) or position 8
to base pair position 359 of the P2B gene in C. albicans
(AF317662.1). C. albicans.
[0033] Particularly preferred, are kits comprising, a probe for a
portion of the P2A C. albicans gene and/or a probe for a portion of
the region of the gene equivalent to base pair position 114 to by
position 439 of C. albicans (AF317661.1).
[0034] Particularly preferred, are kits comprising, a probe for a
portion of the P2B C. albicans gene and/or a probe for a portion of
the region of the gene equivalent to base pair position 1 to
position 359. Also preferred are probes for a portion of the region
of the gene equivalent to base pair position 1 to position 335 in
C. albicans (XM.sub.--718047.1, XM_717900.1) or position 8 to by
position 359 of the P2B gene in C. albicans (AF317662.1).
Equivalent base pair positioning may be found in other organisms,
but not necessarily in the same position.
[0035] The portion of the P2B gene may be equivalent to a portion
of the region of the gene from base pair 24 to by position 158 in
C. glabrata (XM.sub.--444905.1). Particularly preferred, are kits
comprising, a probe for a portion of the P2B C. glabrata gene
and/or a probe for a portion of the region of the gene equivalent
to base pair position 24 to base pair position 158 in C. glabrata.
Sequences equivalent to base pair position 24 to base pair position
158 can be found in other organisms, but not necessarily in the
same position. The kit may also comprise additional primers or
probes.
[0036] The portion of the P2 gene may be equivalent to a portion of
the region of the gene from base pair positions 1 to 326 in A.
fumigatus. Particularly preferred, are kits comprising, a probe for
a portion of the P2 A. fumigatus gene and/or a probe for a portion
of the region of the gene equivalent to base pair position 1 to
base pair position 326 in A. fumigatus. Equivalent sequences to
base pair position 1 to base pair position 326 can be found in
other organisms, but not necessarily in the same position. The kit
may also comprise additional primers or probes.
[0037] The primer may have a sequence selected from the group SEQ
ID NO 1-8, 85-90, 95-104, and a sequence substantially homologous
to or substantially complementary to those sequences, which can
also act as a primer for the P2, P2A and P2B genes. The primers may
also be a primer which preferentially hybridizes to the same
nucleotide sequence as is preferentially hybridized by the primers
SEQ ID NO 1-8, 85-90, 95-104.
[0038] The kit may comprise at least one forward in vitro
amplification primer and at least one reverse in vitro
amplification primer, the forward amplification primer having a
sequence selected from the group consisting of SEQ ID NO 1, 3, 7;
85-88, 95-99, and a sequence being substantially homologous or
complementary thereto which can also act as a forward amplification
primer for the P2A, P2B or P2 genes, and the reverse amplification
primer having a sequence selected from the group consisting of SEQ
ID NO 2, 4, 5, 6, 8, 89, 90, 100-104, and a sequence being
substantially homologous or complementary thereto which can also
act as a reverse amplification primer for the P2A, P2B or P2
genes
[0039] The diagnostic kit may be based on direct nucleic acid
detection technologies, signal amplification nucleic acid detection
technologies, and nucleic acid in vitro amplification technologies
is selected from one or more of Polymerase Chain Reaction (PCR),
Ligase Chain Reaction (LCR), Nucleic Acids Sequence Based
Amplification (NASBA), Strand Displacement Amplification (SDA),
Transcription Mediated Amplification (TMA), Branched DNA technology
(bDNA) and Rolling Circle Amplification Technology (RCAT)), or
other in vitro enzymatic amplification technologies.
[0040] The invention also provides a nucleic acid molecule selected
from the group consisting of SEQ ID NO.1 to SEQ ID NO. 110 and
sequences substantially homologous thereto, or substantially
complementary to a portion thereof and having a function in
diagnostics based on the P2 and/or P2A and P2B genes. The nucleic
acid molecule may comprise an oligonucleotide having a sequence
substantially homologous to or substantially complementary to a
portion of a nucleic acid molecule of SEQ ID NO.1 to SEQ ID NO.
110. The invention also provides a method of detecting a target
organism in a test sample comprising the steps of:
[0041] (i) Mixing the test sample with at least one oligonucleotide
probe as defined above under appropriate conditions; and
[0042] (ii) hybridizing under high stringency conditions any
nucleic acid that may be present in the test sample with the
oligonucleotide to form a probe:target duplex; and
[0043] (iii) determining whether a probe:target duplex is present;
the presence of the duplex positively identifying the presence of
the target organism in the test sample.
[0044] The nucleic acid molecule and kits of the present invention
may be used in a diagnostic assay to detect the presence of one or
more yeast and/or fungal species, to measure yeast and/or fungal
titres in a patient or in a method of assessing the efficacy of a
treatment regime designed to reduce yeast and/or fungal titre in a
patient or to measure yeast and/or fungal contamination in an
environment. The environment may be a hospital, or it may be a food
sample, an environmental sample e.g. water, an industrial sample
such as an in-process sample or an end product requiring bioburden
or quality assessment. The kits and the nucleic acid molecule of
the invention may be used in the identification and/or
characterization of one or more disruptive agents that can be used
to disrupt the P2A, P2B or P2 gene function. The disruptive agent
may be selected from the group consisting of antisense RNA, PNA,
and siRNA.
[0045] In some embodiments of the invention, a nucleic acid
molecule comprising a species-specific probe can be used to
discriminate between species of the same genus. The
oligonucleotides of the invention may be provided in a composition
for detecting the nucleic acids of yeast and fungal target
organisms. Such a composition may also comprise buffers, enzymes,
detergents, salts and so on, as appropriate to the intended use of
the compositions. It is also envisioned that the compositions, kits
and methods of the invention, while described herein as comprising
at least one synthetic oligonucleotide, may also comprise natural
oligonucleotides with substantially the same sequences as the
synthetic nucleotide fragments in place of, or alongside synthetic
oligonucleotides.
[0046] The invention also provides for an in vitro amplification
diagnostic kit for a target yeast and/or fungal organism comprising
at least one forward in vitro amplification primer and at least one
reverse in vitro amplification primer, the forward amplification
primer being selected from the group consisting of one or more of a
sequence being substantially homologous or complementary thereto
which can also act as a forward amplification primer, and the
reverse amplification primer being selected from the group
consisting of one or more of or a sequence being substantially
homologous or complementary thereto which can also act as a reverse
amplification primer.
[0047] The invention also provides for a diagnostic kit for
detecting the presence of candidate yeast and/or fungal species,
comprising one or more DNA probes comprising a sequence
substantially complementary to, or substantially homologous to the
sequence of the P2A, P2B or P2 gene of the candidate yeast and/or
fungal species. The present invention also provides for one or more
synthetic oligonucleotides having a nucleotide sequence
substantially homologous to or substantially complementary to one
or more of the group consisting of the P2A, P2B gene or mRNA
transcripts thereof, the yeast and or fungal P2 gene or mRNA
transcript thereof, the yeast P2B gene or mRNA transcript thereof,
one or more of SEQ ID NO 1-SEQ ID NO 110.
[0048] The nucleotide may comprise DNA. The nucleotide may comprise
RNA. The nucleotide may comprise a mixture of DNA, RNA and PNA. The
nucleotide may comprise synthetic nucleotides. The sequences of the
invention (and the sequences relating to the methods, kits
compositions and assays of the invention) may be selected to be
substantially homologous to a portion of the coding region of the
P2, P2A or P2B genes. The gene may be a gene from a target yeast or
fungal organism. The sequences of the invention are preferably
sufficient so as to be able form a probe:target duplex to the
portion of the sequence.
[0049] The invention also provides for a diagnostic kit for a
target yeast or fungal organism comprising an oligonucleotide probe
substantially homologous to or substantially complementary to an
oligonucleotide of the invention (which may be synthetic). It will
be appreciated that sequences suitable for use as in vitro
amplification primers may also be suitable for use as
oligonucleotide probes: while it is preferable that amplification
primers may have a complementary portion of between about 15
nucleotides and about 30 nucleotides (more preferably about
15-about 23, most preferably about 20 to about 23), oligonucleotide
probes of the invention may be any suitable length. The skilled
person will appreciate that different hybridization and or
annealing conditions will be required depending on the length,
nature & structure (eg. Hybridization probe pairs for
LightCycler, Taqman 5' exonuclease probes, hairpin loop structures
etc. and sequence of the oligonucleotide probe selected.
[0050] Kits and assays of the invention may also be provided
wherein the oligonucleotide probe is immobilized on a surface. Such
a surface may be a bead, a membrane, a column, dipstick, a
nanoparticle, the interior surface of a reaction chamber such as
the well of a diagnostic plate or inside of a reaction tube,
capillary or vessel or the like.
[0051] The target yeast or fungal organism may be selected from the
group consisting of C. albicans, C. glabrata, C. krusei, C.
parapsilosis, C. tropicalis C. dubliniensis, C. guilliermondii, C.
norvegiensis, C. famata, C. haemuloni, C. kefyr, C. utilis, C.
viswanathii, A. fumigatus, A. nidulans, A. clavatus, A. niger, A.
terreus, A. flavus, A. versicolor and Neosartorya fischeri.
[0052] Under these circumstances, the amplification primers and
oligonucleotide probes of the invention may be designed to a gene
specific or genus specific region so as to be able to identify one
or more, or most, or substantially all of the desired organisms of
the target organism grouping.
[0053] The test sample may comprise cells of the target yeast
and/or fungal organism. The method may also comprise a step for
releasing nucleic acid from any cells of the target yeast or fungal
organism that may be present in said test sample. Ideally, the test
sample is a lysate of an obtained sample from a patient (such as a
swab, or blood, urine, saliva, a bronchial lavage, dental specimen,
skin specimen, scalp specimen, transplant organ biopsy, stool,
mucus, or discharge sample). The test samples may be a food sample,
a water sample, an environmental sample, an end product, end
product or in-process industrial sample.
[0054] The invention also provides for the use of any one of SEQ ID
NO.1 to SEQ ID NO.110 in a diagnostic assay for the presence of one
or more yeast or fungal species. The species may be selected from
the group consisting of C. albicans, C. glabrata, C. krusei, C.
parapsilosis, C. tropicalis, C. dubliniensis, C. guilliermondii, C.
norvegiensis, C. famata, C. haemuloni, C. kefyr, C. utilis, C.
viswanathii, A. fumigatus, N. fischeri, A. clavatus, A. niger, A.
terreus, A. flavus, A. versicolor and A. nidulans.
[0055] The invention also provides for kits for use in clinical
diagnostics, theranostics, food safety diagnostics, industrial
microbiology diagnostics, environmental monitoring, veterinary
diagnostics, bio-terrorism diagnostics comprising one or more of
the synthetic oligonucleotides of the invention. The kits may also
comprise one or more articles selected from the group consisting of
appropriate sample collecting instruments, reagent containers,
buffers, labelling moieties, solutions, detergents and
supplementary solutions. The invention also provides for use of the
sequences, compositions, nucleotide fragments, assays, and kits of
the invention in theranostics, Food safety diagnostics, Industrial
microbiology diagnostics, Environmental monitoring, Veterinary
diagnostics, Bio-terrorism diagnostics.
[0056] The nucleic acid molecules, composition, kits or methods may
be used in a diagnostic nucleic acid based assay for the detection
of yeast and/or fungal species.
[0057] The nucleic acid molecules, composition, kits or methods may
be used in a diagnostic assay to measure yeast and/or fungal titres
in a patient. The titres may be measured in vitro.
[0058] The nucleic acid molecules, composition, kits or methods may
be used in a method of assessing the efficacy of a treatment regime
designed to reduce yeast and/or fungal titre in a patient
comprising assessing the yeast and/or fungal titre in the patient
(by in vivo methods or in vitro methods) at one or more key stages
of the treatment regime. Suitable key stages may include before
treatment, during treatment and after treatment. The treatment
regime may comprise an antifungal agent, such as a pharmaceutical
drug. The nucleic acid molecules, composition, kits or methods may
be used in a diagnostic assay to measure potential yeast and/or
fungal contamination, for example, in a hospital. The nucleic acid
molecules, composition, kits or methods may be used in the
identification and/or characterization of one or more disruptive
agents that can be used to disrupt the P2, P2A or P2B gene
function. Suitable disruptive agents may be selected from the group
consisting of antisense RNA, PNA, siRNA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] FIG. 1: Primers binding sites (grey highlights or bold text
or bold and underlined) in P2B of Candida albicans.
[0060] FIG. 2: Primers binding sites in P2 of Aspergillus
fumigatus. The amplified region of interest is underlined (Position
1 to 326).
[0061] FIG. 3: Binding site of C. glabrata probe P1-CglabP2B (bold
and underlined) in the amplified fragment of P2B. PCR primers
CglabP2B-F/CglabP2B-R are highlighted.
[0062] FIG. 4: Binding site of the A. fumigatus probe P1-AspP2
(bold and underlined) in the amplified fragment of P2B. PCR primers
AspP2-F/AspP2-R are highlighted.
[0063] FIG. 5: Resulting amplification curve from Real-Time PCR
assay based on the P2B gene for C. glabrata with P1-CglabP2B probe.
Specificity of the assay was tested using a panel of DNA from 4
other Candida species and Aspergillus fumigatus. The 3 C. glabrata
strains tested were detected and no cross-reaction was observed
with DNA from other species.
[0064] FIG. 6. Resulting amplification curve from Real-time PCR
based on the P2 gene for A. fumigatus with the P1-AspP2 probe.
Specificity of the assay was tested against a panel of DNA from 6
closely related Aspergillus species and C. albicans. The assay
detected A. fumigatus but did not detect or cross-react with DNA
from C. albicans or significantly with any of the Aspergillus
species tested.
[0065] FIG. 7. Alignment of sequence information for Aspergillus
species.
DETAILED DESCRIPTION OF THE INVENTION
Materials and Methods
Cell Culture
[0066] Candida species were cultured in Sabouraud broth (4% wt/vol
glucose, 1% wt/vol peptone, 1.5% agar) for 48 hours at 37.degree.
C. in a shaking incubator. Aspergillus species were cultured in
Sabouraud broth (4% wt/vol glucose, 1% wt/vol peptone, 1.5% agar)
or agar for 3-4 days at 25.degree. C.
DNA Extraction
[0067] Cells from Candida and Aspergillus spp. were pretreated with
lyticase or zymolase enzymes prior to DNA isolation. DNA was
isolated Candida and Apergillus spp. using the MagNA Pure System
(Roche Molecular Systems) in combination with the MagNA pure Yeast
and Bacterial isolation kit III.
DNA sequencing of P2B/P2 gene regions in Candida and Aspergillus
spp.
[0068] The available sequences of the P2B genes of Candida and P2
genes of Aspergillus spp. were acquired from the NCBI database and
aligned using Clustal W. Three annotated sequences for P2B of C.
albicans (XM.sub.--718047.1, XM.sub.--717900.1 and AF317662.1) are
available in the NCBI database and a sequence of high homology to
P2B in C. albicans has been deposited for Candida glabrata
(hypothetical protein, XM.sub.--444905.1), which is considered here
to be P2B. In addition to available sequences for P2 in Aspergillus
spp., three presumptive P2 sequences are deposited as hypothetical
proteins for Aspergillus spp. (XM.sub.--001213622.1,
XM.sub.--001397764.1, XM.sub.--658508.1). PCR Primers were designed
(Table 1) and synthesized by an external company, TibMolBiol,
Germany. Primer set CanP2B-F/CanP2B-R was designed to amplify a 335
by region of the P2B in Candida spp. from positions equivalent to
by position 1 to position 335 in C. albicans (XM.sub.--718047.1,
XM.sub.--717900.1) or position 8 to by position 342 of the P2B gene
in C. albicans (AF317662.1). Primer set CanP2B-F/CanP2B-R may also
in selected Candida species amplify a 326 by region of the P2A
gene. Primer set CanP2B-F/CanP2B flR was designed to amplify a 352
by region of the P2B equivalent to by 8 to by 359 in C. albicans
(AF317662.1). Primer set P2B-F/P2BinR1 were designed to amplify a
312 by region of the P2B gene from by position 1 to by position 312
in XM718047.1/XM 717900.1 and from by position 8 to by 319 of
AF317662.1. Primer set CglabP2B-F/CglabP2B-R were designed to
amplify the region in P2B equivalent to by position 24 to by
position 158 in C. glabrata (XM.sub.--444905.1). Primer set
AspP2-F/AspP2-R were designed to amplify a region of P2 in
Aspergillus spp. from base pair positions equivalent to 1 to
position 326 in A. fumigatus (XM.sub.--750250.1). FIGS. 1 and 2
show the positions of the primer set CanP2B-F/CanP2B-R,CanP2B flR
and P2BinR1 in C. albicans and AspP2-F/AspP2-R in A. fumigatus
respectively. These primer sets were used to amplify regions of the
P2B and P2 genes in other Candida and Aspergillus spp. respectively
by conventional PCR on the iCycler BioRad PCR machine or the PTC200
Peltier thermocycler (MJ Research) using the reagents outlined in
Table 2 and the thermocycling conditions described in Table 3 or
modifications thereof. The PCR reaction products purified with
Roche High Pure PCR Product Purification kit or with the ExoSAP-IT
kit (USB) according to the manufacturers' instructions were sent
for sequencing to Sequiserve, Germany and were sequenced using the
forward amplification primer CanP2B-F or AspP2-F. Sequence
information was obtained for P2B gene regions for 8 Candida spp.
(C. albicans, C. dubliniensis, C. glabrata, C. krusei, C.
parapsilosis, C. tropicalis, C. guillermondii, C. lusitanie) and P2
gene regions for 7 Aspergillus species (A. fumigatus, A. nidulans,
A. clavatus, A. niger, A. terreus, A. flavus, A. versicolor) and
Neosartorya fischeri.
TABLE-US-00001 TABLE 1 PCR primers designed for amplification of
P2B gene region in Candida and P2 gene region in Aspergillus spp.
Primer Name Primer Sequence 5' to 3' CglabP2B-F
ATTGTTGACCCAAGGTGGTAAC CglabP2B-R TCGTCCAAGGACTTACCTTCCAA CanP2B-F
ATGAAATACTTAGCTGCTTAC CanP2B-R TAATCGAATAAACCGAAACCCA CanP2B-flR
GCGACAAGCAATTTCTCT CanP2BinR1 ATCATCATCAGATTCTTCTTTG AspP2-F
ATGAAGCACCTCGCCGC AspP2-R AGACCGAAGCCCATGTC P2ForA GAGGAGCGCCT
P2ForB GAGGAGCGCCTC P2ForC GGAGGAGCGCCTC P2ForD TGAGGAGCGCCTC
P2RevA CCGGAGGGAACGGA P2RevB CCGGAGGGAACGG CAP2BF (For)
ACCTCTCCATCAGCTTCTG CAP2BR (Rev) TCAGCAATCAATTCTTGC CGP2BF (For)
ACCTCTGTCTTATCATCTGTCG CKP2BR (Rev) CTCTTCGACGGACTTACC CGP2BF (For)
AAGAAGGTTATCGAATCTGTTG CGP2BR (Rev) TTCGTCCAAGGACTTACC CTP2BF (For)
TCCGCTTTATTGGAACAAGTTG CTP2BR (Rev) TTCTTGCAAGTCTTTACC CPP2BF (For)
TCCTCATTGTTGGAATCCGTTG CPP2BR (Rev) CTCGTTGATGTCTTTACC
TABLE-US-00002 TABLE 2 PCR reagents used to amplify P2B gene region
in Candida and P2 gene region in Aspergillus spp. SAMPLE PCR
Reaction Mix x1 10x Buffer (100 mM Tris HCl, 15 mM MgCl.sub.2, 5
.mu.l 500 mM KCl pH 8.3) dNTP's Mix, Roche (10 mM dNTP) 1 .mu.l
Primer Forward CanP2B-F or AspP2B-F (10 .mu.M) 1 .mu.l Primer
Reverse CanP2B-R CanP2B-flR, 1 .mu.l Can P2BinR or AspP2B-R (10
.mu.M) Polymerase TaqPol, Roche 1 U/.mu.l 1 .mu.l H2O,
Amgen/Accugene 36-39 .mu.l Genomic DNA Template 2-5 .mu.l TOTAL
VOLUME 50 .mu.l
TABLE-US-00003 TABLE 3 PCR reaction conditions used to amplify P2B
gene region in Candida and P2 gene region in Aspergillus spp. PCR
Thermal profile Step Temp Time 1 94.degree. C. 1 min X 35 2
50.degree. C. 1 min 3 72.degree. C. 1 min 4 72.degree. C. 7 min 5
8.degree. C. Hold Lid preheating was ON
TABLE-US-00004 TABLE 4 TaqMan probes (5'-FAM and 3'-BHQ1 labels)
based on the P2B/P2 gene for C. glabrata and A. fumigatus. Probe
Name Probe Sequence 5'to 3' P1-Cglab-P2B
CAAGAAGGTTATCGAATCTGTTGGTATTG P1-AspP2 CCTGCCGCTGCCGGTGCCGCTGCC
P2FumP ACTACAGCTCGAAGATTA P2FlavP ACGTTGAATGATTGAGAC P2NigP
TTGCGATTACAAGATGGAA P2TerrP CTTCGGACTACTGCGATGA CAP2BP
ACCGCTTTATTGGAATCCGTTG CKP2BP ATCCGACAAGTTAGACAAGTTAATC CGP2BP
AGAATCAACGAATTGTTGTCTGC CTP2BP ATCTTCCAAATTAGACTTATTGTTGA CPP2BP
GAAGAATCAAGATTATCTACCTTGTTG
TABLE-US-00005 TABLE 5 Real-time PCR reagents SAMPLE Preparation of
PCR Reaction Mix x1 HybProb mix 10x conc. (LightCycler .RTM.
FastStartDNA 2 .mu.l Master HybProbe Kit) MgCl.sub.2 stock solution
(Final conc. in reaction is 3 mM) 1.6 .mu.l Probe P1-CglabP2B or
P1-AspP2 2 .mu.l Primer Forward CglabP2B-F or AspP2-F 1 .mu.l
Primer Reverse CglabP2B-R or AspP2-R 1 .mu.l H.sub.2O PCR-grade
10.4 .mu.l Genomic DNA Template 2 .mu.l TOTAL VOLUME 20 .mu.l
TABLE-US-00006 TABLE 6 Real-time PCR thermocycling conditions: PCR
Thermal profile Cycle Step Temp Time Activation 1 95.degree. C. 10
min X 50 Amplification 1 95.degree. C. 10 sec 2 62 or 65.degree. C.
20 sec 3 70.degree. C. 10 sec Cooling 1 40.degree. C. Hold The PCR
was performed with LightCycler .RTM. Roche PCR
Results
Primer and Probe Design
[0069] The publicly available sequence information for the P2B gene
in Candida spp. was aligned with the newly generated sequence
information for the P2B gene in Candida spp. and analysed using
bioinformatics tools. The sequence information available for the P2
gene in Aspergillus spp. was aligned with the newly generated
sequence information for the P2 gene in Aspergillus spp. and
analysed using bioinformatics tools. Species-specific probes were
designed based on the compiled P2B and P2 sequence information for
Candida glabrata and Aspergillus fumigatus respectively (Table 4).
FIGS. 3 and 4 show the relative positions of the PCR primers and
TaqMan DNA probes for the amplification and detection of C.
glabrata and A. fumigatus respectively.
Real-time PCR
[0070] These probes were designed as TaqMan probes and demonstrated
for species detection in real-time PCR on the LightCycler using
reagents and thermocycling conditions detailed in Tables 5 and 6.
For the C. glabrata real-time PCR assay based on the P2B gene, PCR
primers CglabP2B-F/CglabP2B-R were combined with TaqMan probe
P1-Cglab-P2B. The specificity of the assay for the detection of C.
glabrata was confirmed by including DNA from a range of closely
related Candida species and A. fumigatus in the C. glabrata
real-time PCR assay. The assay detected C. glabrata but did not
detect or cross-react with DNA from any other Candida species
tested or with A. fumigatus DNA. FIG. 5 shows the results of the C.
glabrata real-time PCR assay and the specificity of the assay for
C. glabrata. For the A. fumigatus real-time PCR assay based on the
P2 gene, PCR primers AspP2-F/AspP2-R were combined with TaqMan
probe P1-AspP2. The specificity of the assay for the detection of
A. fumigatus was confirmed by including DNA from a range of closely
related Aspergillus species and C. albicans in the A. fumigatus
real-time PCR assay. The assay detected A. fumigatus but did not
detect or cross-react with DNA from C. albicans or any of the four
Aspergillus species tested. Two additional Aspergillus spp. did
show a small amount of cross reaction in the A. fumigatus real-time
PCR assay but this can be eliminated with further assay
optimisation. FIG. 6 shows the results of the A. fumigatus
real-time PCR assay.
[0071] In so far as any sequence disclosed herein differs from its
counterpart in the attached sequence listing in PatentIn3.3
software, the sequences within this body of text are to be
considered as the correct version.
[0072] The words "comprises/comprising" and the words
"having/including" when used herein with reference to the present
invention are used to specify the presence of stated features,
integers, steps or components but does not preclude the presence or
addition of one or more other features, integers, steps, components
or groups thereof.
[0073] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
sub-combination.
SEQ IDs
[0074] Sites of probes, oligonucleotides etc. are shown in bold and
underlined. N or x=any nucleotide; w=a/t, m=a/c, r=a/g, k=g/t,
s=c/g, y=c/t, h=a/t/c, v=a/g/c, d=a/g/t, b=g/t/c. In some cases,
specific degeneracy options are indicated in parenthesis: e.g.:
(a/g) is either A or G.
TABLE-US-00007 SEQ ID NO 1: CglabP2B-F ATTGTTGACCCAAGGTGGTAAC SEQ
ID NO 2: CglabP2B-R TCGTCCAAGGACTTACCTTCCAA SEQ ID NO 3: CanP2B-F
ATGAAATACTTAGCTGCTTAC SEQ ID NO 4: CanP2B-R TAATCGAATAAACCGAAACCCA
SEQ ID NO 5: Can P2BflR1 GCGACAAGCAATTTCTCT SEQ ID NO 6: Can
P2BinR1 ATCATCATCAGATTCTTCTTTG SEQ ID NO 7: AspP2-F
ATGAAGCACCTCGCCGC SEQ ID NO 8: AspP2-R AGACCGAAGCCCATGTC SEQ ID NO
9: P1-Cglab-P2B CAAGAAGGTTATCGAATCTGTTGGTATTG SEQ ID NO 10:
P1-AspP2 CCTGCCGCTGCCGGTGCCGCTGCC SEQ ID NO 11: >glab2897\(PF)
sequences generated for C.glabrata
CTATTGTTGACCCAAGGTGGTAACGAATCTCCAGCTGCTGCTGACATC
AAGAAGGTTATCGAATCTGTTGGTATTGAAGCTGACGAAGCCAGAATC
AACGAATTGTTGTCTGCTTTGGAAGGTAAGTCCTTGGACGAATTGATC
GCTGAAGGTCAACAAAAGTTCGCCTCTGTTCCAGTTGGTGGTGCTGCT
GCTGGTGGTGCTTCCGCTGCTGCTGGTGGTGCCGCTGCCGGTGAAGCC
GCTGAAGAAAAGGAAGAAGAAGCTGCTGAAGAATCCGATGACGACATG
GGTTTCGGTTTATTCGATTA SEQ ID NO 12: >glab8018\(PF) sequences
generated for C.glabrata
TTGTTGACCCAAGGTGGTAACGAATCTCCAGCTGCTGCTGACATCAAG
AAGGTTATCGAATCTGTTGGTATTGAAGCTGACGAAGCCAGAATCAAC
GAATTGTTGTCTGCTTTGGAAGGTAAGTCCTTGGACGAATTGATCGCT
GAAGGTCAACAAAAGTTCGCCTCTGTTCCAGTTGGTGGTGCTGCTGCT
GGTGGTGCTTCCGCTGCTGCTGGTGGTGCCGCTGCCGGTGAAGCCGCT
GAAGAAAAGGAAGAAGAAGCTGCTGAAGAATCCGATGACGACATGGGT TTCGGTTTATTCGATTA
SEQ ID NO 13: >glab4692\(PF) sequences generated for C.glabrata
CTATTGTTGACCCAAGGTGGTAACGAATCTCCAGCTGCTGCTGACATC
AAGAAGGTTATCGAATCTGTTGGTATTGAAGCTGACGAAGCCAGAATC
AACGAATTGTTGTCTGCTTTGGAAGGTAAGTCCTTGGACGAATTGATC
GCTGAAGGTCAACAAAAGTTCGCCTCTGTTCCAGTTGGTGGTGCTGCT
GCTGGTGGTGCTTCCGCTGCTGCTGGTGGTGCCGCTGCCGGTGAAGCC
GCTGAAGAAAAGGAAGAAGAAGCTGCTGAAGAATCCGATGACGACATG
GGTTTCGGTTTATTCGATTA SEQ ID NO 14: >glabl38\(PF) sequences
generated for C.glabrata
CTATTGTTGACCCAAGGTGGTAACGAATCTCCAGCTGCTGCTGACATC
AAGAAGGTTATCGAATCTGTTGGTATTGAAGCTGACGAAGCTAGAATC
AACGAATTGTTGTCTGCTTTGGAAGGTAAGTCCTTGGACGAATTGATC
GCTGAAGGTCAACAAAAGTTCGCCTCTGTTCCAGTTGGTGGTGCTGCT
GCTGGTGGTGCTTCCGCTGCTGCTGGTGGTGCCGCTGCCGGTGAAGCT
GCTGAAGAAAAGGAAGAAGAAGCTGCTGAAGAATCCGATGACGACATG
GGTTTCGGTTTATTCGATTA SEQ ID NO 15: >krusei3165\(P2BF) sequence
generated for C. krusei
TTACTCTTAGTCAATGCTGGTAAAACCGCACCATCTGCTGCAGATGTT
ACCTCTGTCTTATCATCTGTCGGTATCGAAGTTGAATCCGACAAGTTA
GACAAGTTAATCTCCGAATTAGAAGGTAAGTCCGTCGAAGAGTTGATT
GCTGAAGGTACTGAAAAGATGGCTTCTGCTCCAGGTGCAGCAGCTGCT
CCAGCTTCTGGTGCAGGTGCTTCCACCGAATCTGCTGCAGCAGAAGAA
GTTGAAGAAGAAAAGGAAGAATCCGATGATGACATGGGTTTCGGTTTA TTCGATTA SEQ ID NO
16: >krusei9560\(P2BF) sequence generated for C. krusei
CATCTGCTGCAGATGTTACCTCTGTCTTATCATCTGTCGGTATCGAAG
TTGAATCCGACAAGTTAGACAAGTTAATCTCCGAATTAGAAGGTAAGT
CCGTCGAAGAGTTGATTGCTGAAGGTACTGAAAAGATGGCTTCTGCTC
CAGGTGCAGCAGCTGCTCCAGCTTCTGGTGCAGGTGCTTCCACCGAAT
CTGCTGCAGCAGAAGAAGTTGAAGAAGAAAAGGAAGAATCCGATGATG
ACATGGGTTTCGGTTTATTCGATTA SEQ ID NO 17: >krusei60554P2BF)
sequence generated for C. krusei
CATCTGCTGCAGATGTTACCTCTGTCTTATCATCTGTCGGTATCGAAG
TTGAATCCGACAAGTTAGACAAGTTAATCTCCGAATTAGAAGGTAAGT
CCGTCGAAGAGTTGATTGCTGAAGGTACTGAAAAGATGGCTTCTGCTC
CAGGTGCAGCAGCTGCTCCAGCTTCTGGTGCAGGTGCTTCCACCGAAT
CTGCTGCAGCAGAAGAAGTTGAAGAAGAAAAGGAAGAATCCGATGATG
ACATGGGTTTCGGTTTATTCGATTA SEQ ID NO 18: >krusei573\(P2B)
sequence generated for C. krusei
TTACTCTTAGTACATGCTGGTAAAACCGCACCATCTGCTGCAGATGTT
ACCTCTGTCTTATCATCTGTCGGTATCGAAGTTGAATCCGACAAGTTA
GACAAGTTAATCTCCGAATTAGAAGGTAAGTCCGTCGAAGAGTTGATT
GCTGAAGGTACTGAAAAGATGGCTTCTGCTCCAGGTGCAGCAGCTGCT
CCAGCTTCTGGTGCAGGTGCTTCCACCGAATCTGCTGCAGCAGAAGAA
GTTGAAGAAGAAAAGGAAGAATCCGATGATGACATGGGTTTCGGTTTA TTCGATTA SEQ ID NO
19: >CA562P2B sequence generated for C.albicans
TTATTGTTAGTTCAAGGTGGTAACACCTCTCCATCAGCTTCTGATATC
ACCGCTTTATTGGAATCCGTTGGTGTTGAAGCCGAAGAATCCAGATTA
CAAGCTTTATTGAAAGATTTGGAAGGTAAAGACTTGCAAGAATTGATT
GCTGAAGGTAACACCAAATTAGCTTCTGTCCCATCCGGTGGTGCTGCT
GCTGGTGGTGCTTCTGCCTCTGCTGGTGCCGCTGCTGGTGGTGCTGCT
GAAGCTGAAGAAGAAAAAGAAGAAGAAGCCAAAGAAGAATCTGATGAT
GATATGGGTTTCGGTTTATTCGATTAGAGAAATTGCTTGTCGC SEQ ID NO 20:
>A765\(P2BF) P2B sequence generated for C.albicans
TTATTGTTAGTTCAAGGTGGTAACACCTCTCCATCAGCTTCTGATATC
ACCGCTTTATTGGAATCCGTTGGTGTTGAAGCCGAAGAATCCAGATTA
CAAGCTTTATTGAAAGATTTGGAAGGTAAAGACTTGCAAGAATTGATT
GCTGAAGGTAACACCAAATTAGCTTCTGTCCCATCCGGTGGTGCTGCT
GCTGGTGGTGCTTCTGCCTCTGCTGGTGCCGCTGCTGGTGGTGCTGCT
GAAGCTGAAGAAGAAAAAGAAGAAGAAGCCAAAGAAGAATCTGATGAT
GATATGGGTTTCGGTTTATTCGATTAGAGAAATTGCTTGTCGC SEQ ID NO 21:
>A3156\(P2BF) P2B sequence generated for C.albicans
TTATTGTTAGTTCAAGGTGGTAACACCTCTCCATCAGCTTCTGATATC
ACCGCTTTATTGGAATCCGTTGGTGTTGAAGCCGAAGAATCCAGATTA
CAAGCTTTATTGAAAGATTTGGAAGGTAAAGACTTGCAAGAATTGATT
GCTGAAGGTAACACCAAATTAGCTTCTGTCCCATCCGGTGGTGCTGCT
GCTGGTGGTGCTTCTGCCTCTGCTGGTGCCGCTGCTGGTGGTGCTGCT
GAAGCTGAAGAAGAAAAAGAAGAAGAAGCCAAAGAAGAATCTGATGAT
GATATGGGTTTCGGTTTATTCGATTAGAGAAATTGCTTGTCGC SEQ ID NO 22:
>A2700\(P2BF) P2B sequence generated for C.albicans
TTATTGTTAGTTCAAGGTGGTAACACCTCTCCATCAGCTTCTGATATC
ACCGCTTTATTGGAATCCGTTGGTGTTGAAGCYGAAGAATCCAGATTA
CAAGCTTTATTGAAAGATTTGGAAGGTAAAGACTTGCAAGAATTGATT
GCTGAAGGTAACACCAAATTAGCTTCTGTCCCATCCGGTGGTGCTGCT
GCTGGTGGTGCTTCTGCCTCTRCTGGTGCCGCTGCTGGYGGTGCTGCY
GAAGCTGAAGAAGAAAAAGAAGAAGAAGCCAAAGAAGAATCTGATGAT
GATATGGGTTTCGGTTTATTCGATTAGAGAAATTGCTTGTCGC SEQ ID NO 23:
>D3949\(P2BF) P2B sequence generated for C.dubliniensis
TTGTTGTTAGTTCAAGGTGGTAACGCCACTCCATCAGCTTCTGATATC
AGCGCTGTCTTGGAAACTGTTGGTGTTGAAGCCGAAGAATCCAGATTA
CAAGCTTTATTGAAAGATTTGGAAGGTAAAGATTTGCAAGAATTGATT
GCTGAAGGTAACACCAAATTAGCTTCTGTCCCAACCGGTGGTGCTGCT
GCTGGTGGTGCTTCCGGTTCTGCTGGTGCCGCTTCTGGTGCCGCTGCT
GAAGCTGAAGAAGAAAAAGAAGAAGAAGCTAAAGAAGAATCTGATGAT
GATATGGGTTTCGGTTTATTCGATTAGAGAAATTGCTTGTCGC SEQ ID NO 24:
>D7987\(P2BF) sequence generated for C.dubliniensis
TTGTTGTTAGTTCAAGGTGGTAACGCCACTCCATCAGCTTCTGATATC
AGCGCTGTCTTGGAAACTGTTGGTGTTGAAGCCGAAGAATCCAGATTA
CAAGCTTTATTGAAAGATTTGGAAGGTAAAGATTTGCAAGAATTGATT
GCTGAAGGTAACACCAAATTAGCTTCTGTCCCAACCGGTGGTGCTGCT
GCTGGTGGTGCTTCCGGTTCTGCTGGTGCCGCTTCTGGTGCCGCTGCT
GAAGCTGAAGAAGAAAAAGAAGAAGAAGCCAAAGAAGAATCTGATGAT GATA SEQ ID NO 25:
>CP604\(CanP2B-FOR>><<REV) P2B sequences generated
for C.parapsilosis ATGAAATACTTAGCTGCTTACTTATTATTGGTCCAAGGTGGTAACGCC
TCCCCATCTGCTTCAGACATCTCCTCATTGTTGGAATCCGTTGGTGTT
GAAGTTGAAGAATCAAGATTATCTACCTTGTTGAAAGACTTGGAAGGT
AAAGACATCAACGAGTTGATTGCTGAAGGTAACACCAAATTGGCCTCA
GTTCCATCTGGTGGTGCTGCTGTTGCTTCCGGTTCTGGTGCTTCTGGT
GCCGCTGCTGGTGGTGCTGCTGAAGAAGCTAAGGAAGAAGCCAAGGAA
GAAGAAAAGGAAGAATCTGATGATGACATGGGTTTCGGTTTATTCGAT TA SEQ ID NO 26:
>P2194\(P2BF) sequences generated for C.parapsilosis
TTATTATTGGTCCAAGGTGGTAACGCCTCCCCATCTGCTTCAGACATC
TCCTCATTGTTGGAATCCGTTGGTGTTGAAGTTGAAGAATCAAGATTA
TCTACCTTGTTGAAAGACTTGGAAGGTAAAGACATCAACGAGTTGATT
GCTGAAGGTAACACCAAATTGGCCTCAGTTCCATCTGGTGGTGCTGCT
GTTGCTTCCGGTTCTGGTGCTTCTGGTGCCGCTGCTGGTGGTGCTGCT
GAAGAAGCTAAGGAAGAAGCCAAGGAAGAAGAAAAGGAAGAATCTGAT
GATGACATGGGTTTCGGTTTATTCGATTA SEQ ID NO 27: >P96141\(P2BF)
sequences generated for C.parapsilosis
GGTGGTAACGCCTCCCCATCCGCTTCAGACATCTCATCCTTGTTGGAA
TCCGTTGGTGTTGAAGTTGAAGAATCAAGATTGTCCCTCTTGTTGAAA
GACTTGGAAGGTAAAGACATCAACGAATTGATTGCTGAAGGTAACACC
AAGTTGGCTTCAGTTCCAACTGGTGGTGCTGCTGTTGCTTCTGGTTCT
GGTGCTTCAGGTGCCGCTGCTGGTGGTGCTGCTGAAGAAGCCAAAGAA GAATCTGATGATGAT
SEQ ID NO 28: >CT94\(P2BF) sequences generated for C.tropicalis
GGTGGTAACGCTTCCCCATCTGCTTCTGACATCTCCGCTTTATTGGAA
CAAGTTGGTGCTGAAGTTGAATCTTCCAAATTAGACTTATTGTTGAAA
GAATTGGAAGGTAAAGACTTGCAAGAATTGATTGCCGAAGGTAACACT
AAATTCGCCTCTGTCCCATCCGGTGGTGCTGCTGCTGCTTCYTCTGGT
TCCGCTGCCGCTGCTGGTGGTGCCGCTGCCGAAGCTGAAGAAGAAAAA
GAAGAAGAAGCCAAAGAAGAATCTGATGATGAT SEQ ID NO 29: >CT2311\(P2BF)
sequences generated for C.tropicalis
GGTGGTAACGCTTCCCCATCTGCTTCTGACATCTCCGCTTTATTGGAA
CAAGTTGGTGCTGAAGTTGAATCTTCCAAATTAGACTTATTGTTGAAA
GAATTGGAAGGTAAAGACTTGCAAGAATTGATTGCCGAAGGTAACACT
AAATTCGCCTCTGTCCCATCCGGTGGTGCTGCTGCTGCTTCCTCTGGT
TCCGCTGCCGCTGCTGGTGGTGCCGCTGCCGAAGCTGAAGAAGAAAAA
GAAGAAGAAGCCAAAGAAGAATCTGATGATGAT SEQ ID NO 30: >T-8157\(PF)
sequences generated for C.tropicalis
TTATTATTAGTCCAAGGTGGTAACRCTTCCCCATCTGCTTCTGACATC
TCCGCTTTATTGGAACAAGTTGGTGCTGAAGTTGAATCTTCCAAATTA
GACTTATTGTTGAAAGAATTGGAAGGTAAAGACTTGCAAGAATTGATT
GCCGAAGGTAACACTAAATTCGCCTCTGTCCCATCCGGTGGTGCTGCY
GCTGCTTCCTCTGGTTCCGCTGCCGCTGCTGGTGGTGCCGCTGCCGAA
GCTGAAGAAGAAAAAGAAGAAGAAGCCAAAGAAGAATCTGATGATGAT SEQ ID NO 31:
>T-2424\(PF) sequences generated for C.tropicalis
TTATTATTAGTCCAAGGTGGTAACGCTTCCCCATCTGCTTCTGACATC
TCCGCTTTATTGGAACAAGTTGGTGCTGAAGTTGAATCTTCCAAATTA
GACTTATTGTTGAAAGAATTGGAAGGTAAAGACTTGCAAGAATTGATT
GCCGAAGGTAACACTAAATTCGCCTCTGTCCCATCCGGTGGTGCTGCT
GCTGCTTCCTCTGGTTCCGCTGCCGCTGCTGGTGGTGCCGCTGCCGAA
GCTGAAGAAGAAAAAGAAGAAGAAGCCAAAGAAGAATCTGATGATGAT SEQ ID NO 32:
>T-3895\(PF) sequences generated for C.tropicalis
TTATTATTAGTCCAAGGTGGTAACGCTTCCCCATCTGCTTCTGACATC
TCCGCTTTATTGGAACAAGTTGGTGCTGAAGTTGAATCTTCCAAATTA
GACTTATTGTTGAAAGAATTGGAAGGTAAAGACTTGCAAGAATTGATT
GCCGAAGGTAACACTAAATTCGCCTCTGTCCCATCCGGTGGTGCTGCT
GCTGCTTCTTCTGGTTCCGCTGCCGCTGCTGGTGGTGCCGCTGCCGAA
GCTGAAGAAGAAAAAGAAGAAGAAGCCAAAGAAGAATCTGATGATGAT SEQ ID NO 33:
>guill2672\(P2BF) sequences generated for C.guilliermondii
TTRTTGTTGGTKRACGCCGGTAACACCTCCCCATCTGCTGCTGACATC
AAGGCTGTCTTGGAGTCGGTTTCCATAGAAGTTGACGACGAGAAGGTG
TCCAAGTTGTTGAGCGAAGTTGAGGGAAAGAATGCTGAAGAATTGATC
GCTGAAGGTAACGAAAAATTGTCTTCTGTTCCAACTGGTGGACCAGCT
GCTGCTTCCTCTGGATCTGCTGCCGCTGCCGATGCTCCTGCTGCCGAA
GAGGCCGCTGAGGAGGCCGCTGAGGAGTCTGACGACGACATGGGTTTC GGTTTATTCGATTA SEQ
ID NO 34: >guill6021\(P2BF) sequences generated for
C.guilliermondii TTRTTGTTGGTKRACGCCGGTAACACCTCCCCATCTGCTGCTGACATC
AAGGCTGTCTTGGAGTCGGTTTCCATAGAAGTTGACGACGAGAAGGTG
TCCAAGTTGTTGAGCGAAGTTGAGGGAAAGAATGCTGAAGAATTGATC
GCTGAAGGTAACGAAAAATTGTCTTCTGTTCCAACTGGTGGACCAGCT
GCTGCTTCCTCTGGATCTGCTGCCGCTGCCGATGCTCCTGCTGCCGAA
GAGGCCGCTGAGGAGGCCGCTGAGGAGTCTGACGACGACATGGGTTTC GGTTTATTCGATTA SEQ
ID NO 35: >lus7270\(P2BF) sequences generated for C.lusitanie
TTATTGTTGGTCAACGCTGGTAACACCGCCCCATCTGCTGCTGACGTC
AAGAAGGTCTTGGAATCCGTCTCTATTGAGGTTGAGGACGACAAGGTT
GAGAAGTTGTTGGCTGAAGTTGAAGGCAAGAACGTCGAAGAGTTGATT
GCCGAGGGTAACGAGAAGTTGTCTTCTGTTCCATCTGGTGCTCCAGCT
GCTGCTGGTGCCGCTGCTGCTTCTGGTTCTACTGAGGCTGCTGCTGAA
GAGCCACAAGAAGAAGAGAAGGAGGAGTCTGACGACGACATGGGTTTC GGTTTATTCGATTA SEQ
ID NO 36: >AF419.64\(AspP2-F) P2 sequences generated for
A.fumigatus TTACCTCCTCCTCGCCCTTGCTGGCAACACCTCCCCGTCCTCTGAGGA
TGTCAAGGCCGTCCTCTCTTCCGTTGGCATTGATGCCGATGAGGAGCG
CCTGAACAAGCTCATTGCTGAGCTCGAGGGCAAGGACCTCCAGGAGGT
TAGTAACTACAGCTCGAAGATTACAGACTGGGAATTTTGGACTGGCGC
TGACATCGAACTCTACAACAGCTCATTGCCGAGGGTTCCACCAAGCTC
GCTTCCGTTCCCTCCGGTGGTGCTGCCGCCGCTGCTCCTGCCGCTGCC
GGTGCCGCTGCCGGTGGTGCTGCTGCTCCTGCCGCTGAGGAGAAGAAG
GAGGAGGAGAAGGAGGAGTCCGACGAGGACATGGGCTTCGGTCT SEQ ID NO 37:
>F-6951\(APF) P2 sequences generated for A.fumigatus
CCTCCTCCTCGCCCTTGCTGGCAACACCTCCCCGTCCTCTGAGGATGT
CAAGGCCGTCCTCTCTTCCGTTGGCATTGATGCCGATGAGGAGCGCCT
GAACAAGCTCATTGCTGAGCTCGAGGGCAAGGACCTCCAGGAGGTTAG
TAACTACAGCTCGAAGATTACAGACTGGGAATTTTGGACTGGCGCTGA
CATCGAACTCTACAACAGCTCATCGCCGAGGGTTCCACCAAGCTCGCT
TCCGTTCCCTCCGGTGGTGCTGCCGCCGCTGCTCCTGCCGCTGCCGGT
GCCGCTGCCGGTGGTGCTGCTGCTCCTGCCGCTGAGGAGAAGAAGGAG
GAGGAGAAGGAGGAGTCCGACGAGGACATGGGCTTCGGTCT SEQ ID NO 38:
>F-133-61\(APF) P2 sequences generated for A.fumigatus
GCAACACCTCCCCGTCCTCTGAGGATGTCAAGGCCGTCCTCTCTTCCG
TTGGCATTGATGCCGATGAGGAGCGCCTGAACAAGCTCATTGCTGAGC
TCGAGGGCAAGGACCTCCAGGAGGTTAGTAACTACAGCTCGAAGATTA
CAGACTGGGAATTTTGGACTGGCGCTGACATCGAACTCTACAACAGCT
CATTGCCGAGGGTTCCACCAAGCTCGCTTCCGTTCCCTCCGGTGGTGC
TGCCGCCGCTGCTCCTGCCGCTGCCGGTGCCGCTGCCGGTGGTGCTGC
TGCTCCTGCCGCTGAGGAGAAGAAGGAGGAGGAGAAGGAGGAGTCCGA
CGAGGACATGGGCTTCGGTCT SEQ ID NO 39: >AF493.61\(Asp2F) P2
sequences generated for A.fumigatus
TTACCTCCTCCTCGCCCTTGCTGGCAACACCTCCCCGTCCTCTGAGGA
TGTCAAGGCCGTCCTCTCTTCCGTTGGCATTGATGCCGATGAGGAGCG
CCTGAACAAGCTCATTGCTGAGCTCGAGGGCAAGGACCTCCAGGAGGT
TAGTAACTACAGCTCGAAGATTACAGACTGGGAATTTTGGACTGGCGC
TGACATCGAACTCTACAACAGCTCATTGCCGAGGGTTCCACCAAGCTC
GCTTCCGTTCCCTCCGGTGGTGCTGCCGCCGCTGCTCCTGCCGCTGCC
GGTGCCGCTGCCGGTGGTGCTGCTGCTCCTGCCGCTGAGGAGAAGAAG
GAGGAGGAGAAGGAGGAGTCCGACGAGGACATGGGCTTCGGTCT SEQ ID NO 40:
>AF1085-P2\(AspP2-F) P2 sequences generated for N.fischeri
TTACCTCCTCCTCGCCCTTGCTGGCAACACCTCCCCCTCCGCTGAGGA
TGTCAAGGCCGTCCTCTCTTCCGTCGGCATTGACGCCGATGAGGAGCG
CCTGAACAAGCTCATTGCTGAGCTCGAGGGCAAGGACCTCCAGGAGGT
TAGTACACACGGCTTGAATATTACCGACTGAGAATTTTGGACCGGCGC
TGACATCGATTTCTACAACAGCTGATCGCTGAGGGTTCCGCCAAGCTC
GCTTCCGTTCCCTCCGGTGGTGCCGGTGGTGCCGCTGCTCCTGCCGCT
GGCGGTGCCGCTGCCGGTGGTGCTGCTGCCGCTCCCGCCGAAGAGAAG
GAGGAGGAGAAGGAGGAGTCCGACGAGGACATGGGCTTCGGTCT SEQ ID NO 41:
>Nid100-2\(P2F) P2B sequence for A. nidulans
CTACCTCCTCCTCGCCCTTGCTGGCAACGAGTCTCCCTCCGCCTCCGA
CATCAAGGAGGTTCTCTCTTCCGTCGGTGTTGACGCCGACGACGAGCG
CCTCGAGAAGCTCATTGCTGAGCTCCAGGGCAAGGACATCAACGAGGT
TCGTTATTGCATATAGAGTTGGAAGACGCGGACTGCGGGCTAACGATA
ATCTTTAAACAGCTGATCGCTGAGGGTACCACCAAGCTTGCCTCCGTT
CCCTCCGGCGGTGCTGGTGGTGCTGCCCCTGCTGCCGCTGCCGGTGGT
GCTGCTGCTGCCGAGGCCCCCGCTGCTGAGAAGGAGGAGGAGAAGGAG
GAGTCCGATGAGGACATGGGCTTCGGTCT SEQ ID NO 42: >Nid7063\(P2F) P2B
sequence for A. nidulans
CCTTGCTGGCAACGAGTCTCCCTCCGCCTCCGACATCAAGGAGGTTCT
CTCTTCCGTCGGTGTTGACGCCGACGACGAGCGCCTCGAGAAGCTCAT
TGCTGAGCTCCAGGGCAAGGACATCAACGAGGTTCGTTATTGCATAGG
GTTGGAAGACGCGGACAGCGGGCTAACGATAATCTTCTGAACAGCTGA
TCGCTGAGGGTACCACCAAGCTTGCCTCCGTTCCCACCGGCGGTGCTG
GTGCTGCTGCCCCTGCTGCCGCTGCCGGTGGTGCTGCCGCTGCCGAGG
CTCCCGCTGCTGAGAAGGAGGAGGAGAAGGAGGAGTCCGATGAGGACA TGGGCTTCGGTCT SEQ
ID NO 43: >AC5138\(AspP2-F) P2 sequences generated for
A.clavatus TTACCTCCTCCTCGCCCTTGGTGGCAACGCCAGCCCCTCCGCTGCTGA
TGTTAAGGAGGTTCTCTCTTCTGTCGGCATTGATGCTGATGAGGAGCG
CCTCAACAAGCTCATTGCTGAGCTCGAGGGCAAGGACCTTCAGGAGGT
TAGTTTTGCGCTGGTCTACGAGAGGAAGATTGTGACAAGATGCTAACG
GAAAATTTCTTCAACAGCTGATTGCTGAGGGTTCCACCAAGCTCGCTT
CCATTCCCTCCGGCGGTGCTGGTGGTGCTGCCCCCGCCGCTGGCGGTG
CTGCCGCCGGTGGTGCTGCTGAGGCCGCTCCCGCTGAGGAGAAGGAGG
AGGAGAAGGAGGAGTCCGACGACGACATGGGCTTCGGTCT SEQ ID NO 44:
>AN1329399-P2\(AspP2-F) P2 sequences generated for A.niger
CTACCTTCTGTTGGCCCTTGCTGGCAACAACACCCCCTCCGCTGAGGA
CATCAAGTCCGTCCTCTCCGCCGTCGGCATTGACGCTGAGGAGGAGCG
CCTCCAGAAGCTCCTTGCTGAGCTTGAGGGCAAGGACCTCCAGGAGGT
CAGTTAACGCCCTTAAAATCTACCAAGAAATTTTGCGATTACAAGATG
GAATACTGACAATGGGTTTTCTACAACAGCTCATCTCCGAGGGTACCC
AGAAGCTCGCTTCCGTTCCCTCCGGTGGTGCCGGTGCTGCTGCCGCTG
CCCCCGCTGCCGGTGGCGCCGCTGCTGCTGAGGCTCCCGCTGAGGAGA
AGAAGGAGGAGGCTGCTGAGGAGTCCGATGAGGACATGGGCTTCGGTC T SEQ ID NO 45:
>AN2864\(Asp2F) sequences generated for A.niger
CTACCTTCTGTTGGCCCTTGCTGGCAACAACACCCCCTCCGCTGAGGA
CATCAAGTCCGTCCTCTCCGCCGTCGGCATTGACGCTGAGGAGGAGCG
CCTCCAGAAGCTCCTTGCTGAGCTTGAGGGCAAGGACCTCCAGGAGGT
CAGTTAACGCCCTTAAAATCTACCAAGAAATTTTGCGATTACAAGATG
GAATACTGACAATGGTTTTTCTACAACAGCTCATCTCCGAGGGTACCC
AGAAGCTCGCTTCCGTTCCCTCCGGTGGTGCCGGTGCTGCTGCCGCTG
CCCCCGCTGCCGGTGGCGCCGCTGCTGCTGAGGCTCCCGCTGAGGAGA
AGAAGGAGGAGGCTGCTGAGGAGTCCGATGAGGACATGGGCTTCGGTC T SEQ ID NO 46:
>AT118.46\(AspP2 -F) P2 sequences generated for A.terreus
TTCCTTCTCCTCGGCCTTGCCGGCAACACCTCCCCCTCTGCTGAGGAC
ATCAAGGCTGTCCTCTCCTCCGTCGGCATTGACGCTGATGAGGAGCGC
CTCGGCCAGCTCCTGAAGGAGCTCGAGGGCAAGGACATCCAGGAGGTT
AGTGATCACCATAACTTCGGACTACTGCGATGAGAACGCGCCATACTA
ACGGATTATACAGCTCATCGCTCAGGGCTCTGAGAAGCTCGCTTCCGT
TCCCTCTGGCGGTGCTGCCGCTGCTGCTGCTCCCGCCGCTGCCGCTGG
CGGTGACGCTGCTGCCCCCGCTGAGAAGAAGGAGGAGGAGAAGGAGGA
GGAGTCCGACGAGGACATGGGCTTCGGTCT SEQ ID NO 47: >AF2008\(AspP2-F)
P2 sequences generated for A.flavus
TTACCTCCTCCTCGCCCTCGCTGGCAACTCCACCCCCTCCGTTGAGGA
CATCAAGAGCGTTCTCTCTTCCGTCGGTATTGATGCCGATGAGGAGCG
CCTCCAGAAGGTCATCTCCGAGCTCGAGGGCAAGGACCTCCAGCAGGT
TCGTAATACGTTGAATGATTGAGACATAGGGCGCCTGCTGACCATCTA
CTCACTACAGCTGATCACTGAGGGTAGCGAGAAGCTCGCTACCGTTCC
CTCCGGTGGTGCTGGTGCCGCTGCCCCTGCTGCTGGCGGTGCCGCTGC
CGGTGGTGACGCCCCCGCCGCTGAGGAGAAGGAGGAAGAGAAGGAGGA
GTCCGATGAGGACATGGGCTTCGGTCT SEQ ID NO 48: >AV1323\(AspP2-F) P2
sequences generated for A.versicolor
TCGCCCTTGCTGGCAACGAGAGCCCCTCTGCTTCCGACATTAAGGAGG
TTCTGTCCTCCGTCGGTGTTGACGCTGACAACGAGCGCCTCGAGAAGC
TCATCGCTGAGCTCCAGGGCAAGGACATCAACGAGGTTCGTTTTGACA
GATGCATTTGAAATACTTGGCCAGCAGACTAATGAAACCTCTTCTGCA
GTTGATCGCTGAGGGTACCACCAAGCTCGCTTCCGTTCCCTCTGGCGG
TGGTGGTGGTGCTGCCGCCCCCGCTGCTGGTGGCGCTGCCGCCGCTGA
GGCCCCTGCTGCTGAGAAGGAGGAGGAGAAGGAGGAGTCCGACGAGGA ATGGGCTTCGGTCT SEQ
ID NO 49: >AV2916\(Asp2F) sequences generated for A.versicolor
CCCTTGCTGGCAACGAGAGCCCCTCTGCTTCCGACATTAAGGAGGTTC
TGTCCTCCGTCGGTGTTGACGCTGACGACGAGCGCCTCGAGAAGCTCA
TCGCTGAGCTCCAGGGCAAGGACATCAACGAGGTTCGTTTTGACAGAT
GCGTTTGAAATACTTGGCCAGCAGACTAATGAAACCTCTTCTGCAGTT
GATCGCTGAGGGTACCACCAAGCTCGCTTCCGTTCCCTCTGGCGGTGG
TGGTGGTGCTGCCGCCCCCGCTGCTGGTGGCGCTGCCGCCGCTGAGGC
CCCTGCTGCTGAGAAGGAGGAGGAGAAGGAGGAGTCCGACGAGGACAT GGGCTTCGGTCT SEQ
ID NO 50: >Published P2B sequences for C albicans
gi|11229041|gb|AF317662.1|AF317662 Candida albicans 60S acidic
ribosomal protein type P2-B (p2B) gene, complete cds
GGAAAAAATGAAATACTTAGCTGCTTACTTATTGTTAGTTCAAGGTGG
TAACACCTCTCCATCAGCTTCTGATATCACCGCTTTATTGGAATCCGT
TGGTGTTGAAGCCGAAGAATCCAGATTACAAGCTTTATTGAAAGATTT
GGAAGGTAAAGACTTGCAAGAATTGATTGCTGAAGGTAACACCAAATT
AGCTTCTGTCCCATCCGGTGGTGCTGCTGCTGGTGGTGCTTCTGCCTC
TACTGGTGCCGCTGCTGGTGGTGCTGCCGAAGCTGAAGAAGAAAAAGA
AGAAGAAGCCAAAGAAGAATCTGATGATGATATGGGTTTCGGTTTATT
CGATTAGAGAAATTGCTTGTCGCCTTTGCTGGTTTGAGAGAAGTATAT
TTCCATTATTTTGCATTATATATATATATATGTATTATAACTAATCTA
ATAAAAAAAATATGAAAACAAAAATGGCTTCTATATGGCACTGTTTGC A SEQ ID NO 51:
>Published P2B sequences for C.glabrata
gi|50284952|ref|XM_444905.1| Candida glabrata CBS138 hypothetical
protein (CAGLOA03168g) partial mRNA
ATGAAGTACTTGGCCGCTTACCTATTGTTGACCCAAGGTGGTAACGAA
TCTCCAGCTGCTGCTGACATCAAGAAGGTTATCGAATCTGTTGGTATT
GAAGCTGACGAAGCTAGAATCAACGAATTGTTGTCTGCTTTGGAAGGT
AAGTCCTTGGACGAATTGATCGCTGAAGGTCAACAAAAGTTCGCCTCT
GTTCCAGTTGGTGGTGCTGCTGCTGGTGGTGCTTCCGCTGCTGCTGGT
GGTGCCGCTGCCGGTGAAGCTGCTGAAGAAAAGGAAGAAGAAGCTGCT
GAAGAATCCGATGACGACATGGGTTTCGGTTTGTTCGACTAA SEQ ID NO 52:
>gi|68465556|ref|XM_718047.1| Candida albicans SC5314 cytosolic
ribosomal acidic protein P2B (CaO19_5928) partial mRNA
ATGAAATACTTAGCTGCTTACTTATTGTTAGTTCAAGGTGGTAACACC
TCTCCATCAGCTTCTGATATCACCGCTTTATTGGAATCCGTTGGTGTT
GAAGCCGAAGAATCCAGATTACAAGCTTTATTGAAAGATTTGGAAGGT
AAAGACTTGCAAGAATTGATTGCTGAAGGTAACACCAAATTAGCTTCT
GTCCCATCCGGTGGTGCTGCTGCTGGTGGTGCTTCTGCCTCTGCTGGT
GCCGCTGCTGGTGGTGCTGCTGAAGCTGAAGAAGAAAAAGAAGAAGAA
GCCAAAGAAGAATCTGATGATGATATGGGTTTCGGTTTATTCGATTAG SEQ ID NO 53:
>gi|68465849|ref|XM_717900.1| Candida albicans SC5314 cytosolic
ribosomal acidic protein P2B (CaO19_13349) partial mRNA
ATGAAATACTTAGCTGCTTACTTATTGTTAGTTCAAGGTGGTAACACC
TCTCCATCAGCTTCTGATATCACCGCTTTATTGGAATCCGTTGGTGTT
GAAGCCGAAGAATCCAGATTACAAGCTTTATTGAAAGATTTGGAAGGT
AAAGACTTGCAAGAATTGATTGCTGAAGGTAACACCAAATTAGCTTCT
GTCCCATCCGGTGGTGCTGCTGCTGGTGGTGCTTCTGCCTCTGCTGGT
GCCGCTGCTGGTGGTGCTGCTGAAGCTGAAGAAGAAAAAGAAGAAGAA
GCCAAAGAAGAATCTGATGATGATATGGGTTTCGGTTTATTCGATTAG SEQ ID NO 54:
>Published P2 sequence for A.fumigatus
gi|71001323|ref|XM_750250.1| Aspergillus fumigatus Af293 60S acidic
ribosomal protein P2/allergen Asp F 8 (AFUA_2G10100) mRNA, complete
cds ATGAAGCACCTCGCCGCTTACCTCCTCCTCGCCCTTGCTGGCAACACC
TCCCCGTCCTCTGAGGATGTCAAGGCCGTCCTCTCTTCCGTTGGCATT
GATGCCGATGAGGAGCGCCTGAACAAGCTCATTGCTGAGCTCGAGGGC
AAGGACCTCCAGGAGCTCATCGCCGAGGGTTCCACCAAGCTCGCTTCC
GTTCCCTCCGGTGGTGCTGCCGCCGCTGCTCCTGCCGCTGCCGGTGCC
GCTGCCGGTGGTGCTGCTGCTCCTGCCGCTGAGGAGAAGAAGGAGGAG
GAGAAGGAGGAGTCCGACGAGGACATGGGCTTCGGTCTTTTCGACTAA SEQ ID NO 55:
Published P2 sequence for N.fischeri
gi|119480930|ref|1XM_001260493.1|Neosartorya fischeri NRRL 181 60S
acidic ribosomal protein P2/allergen Asp F 8 (NFIA_085510) mRNA,
complete cds ATGAAGCACCTCGCCGCTTACCTCCTCCTCGCCCTTGCTGGCAACACC
TCCCCGTCCGCTGAGGATGTCAAGGCCGTCCTCTCTTCCGTTGGCATT
GACGCCGATGAGGAGCGCCTGAACAAGCTCATTGCTGAGCTCGAGGGC
AAGGACCTCCAGGAGCTCATCGCCGAGGGTTCCACCAAGCTCGCTTCC
GTTCCCTCCGGTGGTGCTGCCGCCGCTGCTCCTGCCGCTGGCGGTGCC
GCTGCCGGTGGTGCTGCTGCTCCTGCCGCTGAGGAGAAGAAGGAGGAG
GAGAAGGAGGAGTCCGACGAGGACATGGGCTTCGGTCTCTTCGACTAA SEQ ID NO 56:
Published P2 sequence for A.clavatus
gi|121715403|ref|XM_001275310.1| Aspergillus clavatus NRRL 1 60S
acidic ribosomal protein P2, putative (ACLA_069130) mRNA, complete
cds ATGAAGCACCTCGCCGCTTACCTCCTCCTCGCCCTTGGTGGCAACGCC
AGCCCCTCCGCTGCTGATGTTAAGGAGGTTCTCTCTTCTGTCGGCATT
GATGCTGATGAGGAGCGCCTCAACAAGCTCATTGCTGAGCTCGAGGGC
AAGGACCTTCAGGAGCTGATTGCTGAGGGTTCCACCAAGCTCGCTTCC
ATTCCCTCCGGCGGTGCTGGTGGTGCTGCCCCCGCCGCTGGCGGTGCT
GCCGCCGGTGGTGCTGCTGAGGCCGCTCCCGCTGAGGAGAAGGAGGAG
GAGAAGGAGGAGTCCGACGACGACATGGGCTTCGGTCTCTTCGACTAA SEQ ID NO 57:
Published P2 sequence for A.terreus gi|115395965|
ref|XM_001213622.1| Aspergillus terreus NIH2624 predicted protein
(ATEG_04444) mRNA, complete cds
ATGAAGCACCTCGCCGCTTTCCTTCTCCTCGGCCTTGCCGGCAACACC
TCCCCCTCTGCTGAGGACATCAAGGCTGTCCTCTCCTCCGTCGGCATT
GACGCTGATGAGGAGCGCCTCGGCCAGCTCCTGAAGGAGCTCGAGGGC
AAGGACATCCAGGAGCTCATCGCTCAGGGCTCTGAGAAGCTCGCCTCC
GTTCCCTCTGGCGGTGCTGCCGCTGGTGCTGCTGCTCCCGCCGCTGCC
GCTGGCGGTGACGCTGCTGCCCCCGCTGAGAAGAAGGAGGAGGAGAAG
GAGGAGGAGTCCGACGAGGACATGGGCTTCGGTCTCTTCGACTAA SEQ ID NO 58:
Published P2 sequence for A. niger gi|145252575|
ref|XM_001397764.1| Aspergillus niger CBS 513.88 hypothetical
protein (An16g04930) mRNA, complete cds
ATGAAGTACCTCGCCGCCTACCTTCTGTTGGCCCTTGCTGGCAACAAC
ACCCCCTCCGCTGAGGACATCAAGTCCGTCCTCTCCGCCGTCGGCATT
GACGCTGAGGAGGAGCGCCTCCAGAAGCTCCTTGCTGAGCTTGAGGGC
AAGGACCTCCAGGAGCTCATCTCCGAGGGTACCCAGAAGCTCGCTTCC
GTTCCCTCCGGTGGTGCCGGTGCTGCTGCCGCTGCCCCCGCTGCCGGT
GGCGCCGCTGCTGCTGAGGCTCCCGCTGAGGAGAAGAAGGAGGAGGCT
GCTGAGGAGTCCGATGAGGACATGGGCTTCGGTCTCTTCGACTAA SEQ ID NO 59:
>gi|67539651|ref|XM_658508.1|Aspergillus nidulans FGSC A4
chromosomel ATGAAGCACCTCGCAGCCTACCTCCTCCTCGCCCTTGCTGGCAACGAG
TCTCCCTCCGCCTCCGACATCAAGGAGGTTCTCTCTTCCGTCGGTGTT
GACGCCGACGACGAGCGCCTCGAGAAGCTCATTGCTGAGCTCCAGGGC
AAGGACATCAACGAGCTGATCGCTGAGGGTACCACCAAGCTTGCCTCC
GTTCCCTCCGGCGGTGCTGGTGGTGCTGCCCCTGCTGCCGCTGCCGGT
GGTGCTGCTGCTGCCGAGGCCCCCGCTGCTGAGAAGGAGGAGGAGAAG
GAGGAGTCCGATGAGGACATGGGCTTCGGTCTCTTCGACTAA SEQ ID NO 60:
>gi|11229039|gb|AF317661.1| Candida albicans 60S acidic
ribosomal protein type P2-A (p2A) gene, complete cds
GAATTCGATTATTGCATTCTGATATTCCCTGCTTTAAATGCATTTGGA
AATATTTCGTATATCATGAGATATAATAACATTAATAGCATTTTCATG
TTACTAACAAGAATATAGTGAAATACTTAGCTGCTTACTTATTATTAG
TTAACGCCGGTAACGCCACCCCATCTGCTGCCGATGTCAAAGCTGTTT
TGTCAGCTGCTGATATTGAAGTCGAAGAAGAAAAAGTTGAAAAATTGA
TCAGCGAATCGGACGGTAAGAACGTCGAAGAATTGATTGCTGAAGGTA
ACGAAAAATTATCATCAGTCCCATCTGGTGCTCCAGCTGCTGCTGCTG
GTGGTGCTTCTGCTGCCGCCGGTGGTGAAGCCACTGAAGAAGCTGCTG
AAGAAGAAGCTGCTGAAGAATCTGATGACGATATGAGTTTCGGTTTAT
TCGATTAAACGAGTCAACAGGCATCTCAAGATCACAGCATAAGG A. fumigatus SEQ ID NO
110: >A. FUM505.62
TTACCTCCTCCTCGCCCTTGCTGGCAACACCTCCCCGTCCTCTGAGGA
TGTCAAGGCCGTCCTCTCTTCCGTTGGCATTGATGCCGATGAGGAGCG
CCTGAACAAGCTCATTGCTGAGCTCGAGGGCAAGGACCTCCAGGAGGT
TAGTAACTACAGCTCGAAGATTACAGACTGGGAATTTTGGACTGGCGC
TGACATCGAACTCTACAACAGCTCATTGCCGAGGGTTCCACCAAGCTC
GCTTCCGTTCCCTCCGGTGGTGCTGCCGCCGCTGCTCCTGCCGCTGCC
GGTGCCGCTGCCGGTGGTGCTGCTGCTCCTGCCGCTGAGGAGAAGAAG
GAGGAGGAGAAGGAGGAGTCCGACGAGGACATGGGCTTCGGTCT A. flavus SEQ ID NO
61: >A.flavus2199\(P2)
TCGCCCTCGCTGGCAACTCCACCCCCTCCGTTGAGGACATCAAGAGCG
TTCTCTCTTCCGTCGGTATTGATGCCGATGAGGAGCGCCTCCAGAAGG
TCATCTCCGAGCTCGAGGGCAAGGACCTCCAGCAGGTTCGTAATACGT
TGAATGATTGAGACATAGGGCGCCTGCTGACCATCTACTCACTACAGC
TGATCACTGAGGGTAGCGAGAAGCTCGCTACCGTTCCCTCCGGTGGTG
CTGGTGCCGCTGCCCCTGCTGCTGGCGGTGCCGCTGCCGGTGGTGACG
CCCCCGCCGCTGAGGAGAAGGAGGAAGAGAAGGAGGAGTCCGATGAGG ACATGGGCTTCGGTCT
A. niger SEQ ID NO 62: >A. nig2828\(P2F)
CTACCTTCTGTTGGCCCTTGCTGGCAACAACACCCCCTCCGCTGAGGA
CATCAAGTCCGTCCTCTCCGCCGTCGGCATTGACGCTGAGGAGGAGCG
CCTCCAGAAGCTCCTTGCTGAGCTTGAGGGCAAGGACCTCCAGGAGGT
CAGTTAACGCCCTTAAAAATCTACCAAGAAATTTTGCGATTACAAGAT
GGAATACTGACAATGGTTTTTTCTACAACAGCTCATCTCCGAGGGTAC
CCAGAAGCTCGCTTCCGTTCCCTCCGGTGGTGCCGGTGCTGCTGCCGC
TGCCCCCGCTGCCGGTGGCGCCGCTGCTGCTGAGGCTCCCGCTGAGGA
GAAGAAGGAGGAGGCTGCTGAGGAGTCCGATGAGGACATGGGCTTCGG TCT A. terreus SEQ
ID NO 63: >A. terr307
TTTCCTTCTCCTCGGCCTTGCCGGCAACACCTCCCCCTCTGCTGAGGA
CATCAAGGCTGTCCTCTCCTCCGTCGGCATTGACGCTGATGAGGAGCG
CCTCGGCCAGCTCCTGAAGGAGCTCGAGGGCAAGGACATCCAGGAGGT
TAGTGATCACCATAACTTCGGACTACTGCGATGAAAACGCGCCATACT
AACGGATTATACAGCTCATCGCTCAGGGCTCTGAGAAGCTCGCCTCCG
TTCCCTCTGGCGGTGCTGCCGCTGGTGCTGCTGCTCCCGCCGCTGCCG
CTGGCGGTGACGCTGCTGCCCCCGCTGAGAAGAAGGAGGAGGAGAAGG
AGGAGGAGTCCGACGAGGACATGGGCTTCGGTCT SEQ ID NO 64: >A. terr5677
TTTCCTTCTCCTCGGCCTTGCCGGCAACACCTCCCCCTCTGCTGAGGA
CATCAAGGCTGTCCTCTCCTCCGTCGGCATTGACGCTGATGAGGAGCG
CCTCGGCCAGCTCCTGAAGGAGCTCGAGGGCAAGGACATCCAGGAGGT
TAGTGATCACCATAACTTCGGACTACTGCGATGAAAACGCGCCATACT
AACGGATTATACAGGTCATCGCTCAGGGCTCTGAGAAGCTCGCCTCCG
TTCCCTCTGGCGGTGCTGCCGCTGGTGCTGCTGCTCCCGCCGCTGCCG
CTGGCGGTGACGCTGCTGCCCCCGCTGAGAAGAAGGAGGAGGAGAAGG
AGGAGGAGTCCGACGAGGACATGGGCTTCGGTCT SEQ ID NO 65: >A. terr2729
TTTCCTTCTCCTCGGCCTTGCCGGCAACACCTCCCCCTCTGCTGAGGA
CATCAAGGCTGTCCTCTCCTCCGTCGGCATTGACGCTGATGAGGAGCG
CCTCGGCCAGCTCCTGAAGGAGCTCGAGGGCAAGGACATCCAGGAGGT
TAGTGATCACCATAACTTCGGACTACTGCGATGAAAACGCGCCATACT
AACGGATTATACAGCTCATCGCTCAGGGCTCTGAGAAGCTCGCCTCCG
TTCCCTCTGGCGGTGCTGCCGCTGGTGCTGCTGCTCCCGCCGCTGCCG
CTGGCGGTGACGCTGCTGCCCCCGCTGAGAAGAAGGAGGAGGAGAAGG
AGGAGGAGTCCGACGAGGACATGGGCTTCGGTCT SEQ ID NO 66: >A. terr601-65
TTTCCTTCTCCTCGGCCTTGCCGGCAACACCTCCCCCTCTGCTGAGGA
CATCAAGGCTGTCCTCTCCTCCGTCGGCATTGACGCTGATGAGGAGCG
CCTCGGCCAGCTCCTGAAGGAGCTCGAGGGCAAGGACATCCAGGAGGT
TAGTGATCACCATAACTTCGGACTACTGCGATGAAAACGCGCCATACT
AACGGATTATACAGCTCATCGCTCAGGGCTCTGAGAAGCTCGCCTCCG
TTCCCTCTGGCGGTGCTGCCGCTGGTGCTGCTGCTCCCGCCGCTGCCG
CTGGCGGTGACGCTGCTGCCCCCGCTGAGAAGAAGGAGGAGGAGAAGG
AGGAGGAGTCCGACGAGGACATGGGCTTCGGTCT A. candidus SEQ ID NO 67: >A.
Cand 225-8\(P2F) TGCTCCTCGGCCTCGCMGGCAACGAGACTCCCTCCGCTGCCGACATCA
AGGGCGTTCTGTCCGCCGTCGGCATTGACGCCGATGAGGACCGTCTCT
CCAAGCTCCTCTCCGAGCTTGAGGGCAAGGACATCAACGAGGTTCGTA
TCTCACAGGAATCGCACACGTAACAGAGTCAACAAATACTAATCCCCC
GTGCAGCTGATCGCCCAGGGCTCCGAGAAGCTTGCTTCCGTTCCCTCC
GGTGGTGCCGCTGGTGGTGCCGCTGCCGCCCCTGCCGCCGCCGCTGGT
GGTGACGCTCCCGCCCAGGAGAAGGAGGAGGAGAAGGAGGAGTCCGAT
GAGGACATGGGCTTCGGTCT SEQ ID NO 68: >A. Cand14607\(P2F)
TGCTCCTCGGCCTCGCCGGCAACGAGTCTCCCTCCGCTGCCGACATCA
AGGGCGTTCTGTCCGCCGTCGGCATTGACGCCGATGAGGAGCGTCTCT
CCAAGCTCCTCTCCGAGCTTGAGGGCAAGGACATCAACGAGGTTCGTA
TCCTGGAATCGCACCGAGACAGAGTCAACAAATACTAATCCCCCGTGC
AGCTGATCGCCCAGGGTACCGAGAAGCTTGCTTCCGTTCCCTCCGGTG
GCGCCGGCGCTGCTGCTGCCGCCCCTGCCGCCGCTGCTGGTGGTGAGG
CCGCCGCTGAGGAGAAGAAGGAGGAGGAGAAGGAGGAGTCCGATGAGG ACATGGGCTTCGGTCT
SEQ ID NO 69: >A. Cand-9695\(APF)
GGCAACCGAGTCTCCCTCCGCTGCCGACATCAAGGGCGTTCTGTCCGC
CGTCGGCATTGACGCCGATGAGGAGCGTCTCTCCAAGCTCCTCTCCGA
GCTTGAGGGCAAGGACATCAACGAGGTTCGTATCCTGGAATCGCACCG
AGACAGAGTCAACAAATACTAATCCCCCGTGCAGCTGATCGCCCAGGG
TACCGAGAAGCTTGCTTCCGTTCCCTCCGGTGGCGCCGGCGCTGCTGC
TGCCGCCCCTGCCGCCGCTGCTGGTGGTGAGGCCGCCGCTGAGGAGAA
GAAGGAGGAGGAGAAGGAGGAGTCCGATGAGGACATGGGCTTCGGTCT A. clavatus SEQ ID
NO 70: >A. clav7944\(P2F)
TTACCTCCTCCTCGCCCTTGGTGGCAACGCCAGCCCCTCCGCTGCTGA
TGTTAAGGAGGTTCTCTCTTCCGTCGGCATTGATGCTGATGAGGAGCG
CCTCAACAAGCTCATTGCTGAGCTCGAGGGCAAGGACCTTCAGGAGGT
TAGTTTTACGCTGGTCTACGAGAGGAAGATTGTGACAAGATGCTAACG
GAAAATTTCTTCAACAGCTGATTGCTGAGGGTTCCACCAAGCTCGCTT
CCATTCCCTCCGGCGGTGCTGGTGGTGCTGCCCCCGCCGCTGGCGGTG
CTGCCGCCGGTGGTGCTGCTGAGGCCGCTCCCGCTGAGGAGAAGGAGG
AGGAGAAGGAGGAGTCCGACGACGACATGGGCTTCGGTCT SEQ ID NO 71: >A.
clav443\(P2F) TTACCTCCTCCTCGCCCTTGGTGGCAACGCCAGCCCCTCCGCTGCTGA
TGTTAAGGAGGTTCTCTCTTCCGTTGGCATTGATGCTGATGAGGAGCG
CCTCAATAAGCTCATTGCTGAGCTTGAGGGCAAGGACCTTCAGGAGGT
TAGTTTACGCCAATCCGCGATATGAAAATTGCGACATGATGCTAACAG
GAGAATTTTTCAACAGCTGATTGCTGAGGGTTCCACCAAGCTCGCTTC
CATTCCTTCCGGCGGTGCTGCCGGTGCTGCCCCTGCCGCTGGCGCTGC
TGCCGGTGGTGCCGCTGAGGCCGCTCCCGCTGAGGAGAAGGAGGAGGA
GAAGGAGGAGTCCGACGACGACATGGGCTTCGGTCT A. glaucus SEQ ID NO 72:
>A. glau2425\(P2F)
ACACGGTGGACTGGCTGGCCGGAAGAGCCGTTGCTGGTGTAAGTTTTG
GTAGGACCGTTCAGCTCCRGAKGTCTTTGGGGACAATGGCGACGAGGG
CTGTCGATCGTTAGTGTACATTCCAAGGAAGGTAAAAGAAAAAGAATG
CGACATACAGTAAGTTGAGCCACTCTGACGCTGGCAGTCATCGCAGTG
GTCATAAGCTGTAGCGAGCGGCTGCTCGATGTTGACCGAGTAGGTGAC
GGCTTTGCAGAGACAGTGACCGGAAATAGGCATTTTGGACGAGAGGCA
GAATATAATTGGACTATAGACTGACAATGATGATGACGATACTGAGGA
CCTTTGGGAGGAATGAAACAGATATTTATACCCTCTCCGACCTCGTGT
AGTCCATCTGTCTTTCGCTGTCTCTCGGTGGACATGGGCTTCGGTCT SEQ ID NO 73:
>A. glau542 GCGTTGGTGCACGACGGTACCAAGGGGACTCTCATACTCCTTAATTCT
CTGGCCGGTCCAGTGTCGCGACTCRTATGGAGCACATTAGCAGATATC
CCACAGAGCTACCATGTCGTCCAGGCTCTTTGTCTGCTTTGCACATGG
CCTTTTCCAACCAGCAGTACCTCCACAGACCCGACGTTCATGCTGTGT
GGTATGATGATGCAAGTCGCCATGCAACTTGGTCTTCACCGGCCTTCG
CACACTCAGGACTTTASCAAGTTCACAGTGGAGCTGATTGAGGAGGAG
CTCAGGGATAAAGTGAGGACATGGGCTTCGGTCT SEQ ID NO 74: >A.
glaucus29771\(P2) GCGTTGGAGCACGACTGTACCAGGATACTCATCATCTCCTTAATTTTC
TGGCCGGTCCAGCGYCGCGACTAGTATGGAGCACATTATCAGACATCC
GACAGAGCTACCATGTCGTAAAGGCTCTTTGTCTGCTCTGCACATGGC
CTTTTCCGACCAGCAGTACCTCCACAGACCCTACKTTTATGCTGTGTG
GTATGATGATGCAAGTCGCCATGCAGCTTGGTCTTCACCGGCCTTCGC
ACACTCAGGACTTTAGCAAGTTTACAGTGGAGCTGATTGAGGAGGAAC
TAAGGGACAAAGTGAGGACATGGGCTTCGGTCT A. versicolor SEQ ID NO 75:
>A.Vers6898 CGCCCTTGCTGGCAACCAGAGCCCCTCTGCTTCCGATGTTAAGGAGGT
TCTCTCCTCCGTCGGTGTTGACGCTGACTCTGAGCGCCTCGAGAAGCT
CATCGCTGAGCTCCAGGGCAAGGACATCAACGAGGTTCGTTTTGACAG
AACCGCTTGAAATTCTTGGCAGCAGACTAATGAAACATTTTCTGCAGT
TGATCGCTGAGGGTACCACTAAGCTCGCTTCCGTTCCCTCTGGCGGTG
CTGGTGCTGCTGCTGCTCCCGCTGCTGGTGGCGCTGCTGCCGCTGAGG
CCCCTGCCGCCGAGAAGGAGGAGGAGAAGGAGGAGTCCGACGAGGACA TGGGCTTCGGTCT SEQ
ID NO 76: >lus66\(P2BF) sequence generated for C. lusitaniae
KTATTGTTGGKCAACGCTGGTAACACCGCCCCATCTGCTGCTGACGTC
AAGAAGGTCTTGGAATCCGTCTCTATTGAGGTTGAGGACGACAAGGTT
GAGAAGTTGTTGGCTGAAGTTGAAGGCAAGAACGTCGAAGAGTTGATT
GCCGAGGGTAACGAGAAGTTGTCTTCTGTTCCATCTGGTGCTCCAGCT
GCTGCTGGTGCCGCTGCTGCTTCTGGTTCTACTGAGGCTGCTGCTGAA
GAGCCACAAGAAGAAGAGAAGGAGGAGTCTGACGACGACATGGGTTTC GGTTTATTCGATTA SEQ
ID NO 77: >fam1\(P2BF) sequence generated for C. famata
CTTCTCCATCCGCTTCTGACATCAGTAGTTTATTAGAAACCGTTGGTG
CTGAAGCTGACGAAGCTAGAATCAGTGCTTTATTGAAGGACTTAGAAG
GTAAGCAAGTCGCTGACTTAATTGCTGAAGGTCAAACCAAGTTGGCTT
CCGTTCCAACTGGTGGTGCTGGTGCTGCTGCTGGTGGTGCCGCTGCTG
CTTCTGGTGATGCCGGTGCAGCTGCTGCTGAAGAAGAAAAGGAAGAAG
AAAAGGAAGAATCCGACGATGACATGGGTTTCGGTTTATTCGATTA SEQ ID NO 78:
>fam2\(P2BF) sequence generated for C. famata
CTTCTCCATCAGCCTCTGACGTCAGTGCTTTATTAGAAACCGTTGGTG
CTGAAGTTGACCAAGGTAGAGTTAGTGCTTTATTGAAGGACTTAGAAG
GTAAGCAAGTTGCCGACTTAATTGCTGAAGGTCAAACCAAGTTAGCTT
CTGTCCCAACCGGTGGTGCTGCTTCTGCTGGTGGTGCTGCCGCTGCTT
CTGGTGCTGCCGGTGCAGCTGCTGTTGAAGAAGAAAAGGAAGAAGAAA
AGGAAGAATCCGATGAAGATATGGGTTTCGGTTTATTCGATT SEQ ID NO 79:
>fam5\(P2BF) sequence generated for C. famata
CTTCTCCATCAGCCTCTGACGTCAGTGCTTTATTAGAAACCGTTGGTG
CTGAAGTTGACCAAGGTAGAGTCAGTGCTTTATTGAAGGACTTAGAAG
GTAAGCAAGTCGCCGACTTAATTGCTGAAGGTCAAACCAAGTTAGCTT
CTGTCCCAACTGGTGGTGCTGCTTCTGCTGGTGGTGCTTCCGCTGCTG
CTTCTGGTGATGCCGGTGCAGCTGCTGCTGAAGAAGAAAAGGAAGAAG
AAAAGGAAGAATCCGATGAAGATATGGGTTTCGGTTTATTCGATTA SEQ ID NO 80:
>haem54\(P2BF) sequence generated for C. haemuloni
TTTCGGTTTATTCGATTAACACTTCCCCAGCTGCCTCTGACATCAAGA
AGGTGTTGGAGTCTGTCTCCATCGAGGTTGAGGACGACAAGGTCGAGA
AGTTGTTGGCTGAGGTCGAGGGCAAGAACGCCGAGGAGTTGATTGCCG
AGGGTAACGAGAAGTTGTCTTCTGTCCCAACTGGTGCTCCAGCTGGTG
GTGCTGCCGCTGCTGGTGGTGCTGCTCCAGAGGCTGCTGCTGAGAAGG
AAGAGGAGGCCGCTGCCGAGGAGTCTGACGACGACATGGGTTTCGGTT TATTCGATTA SEQ ID
NO 81: >haem55\(P2BF) sequence generated for C. haemuloni
TTATTGTTGGTCAACGCCGGTAACACTTCCCCAGCTGCCTCTGACATC
AAGAAGGTGTTGGAGTCTGTCTCCATCGAGGTTGAGGACGACAAGGTC
GAGAAGTTGTTGGCTGAGGTCGAGGGCAAGAACGCCGAGGAGTTGATT
GCCGAGGGTAACGAGAAGTTGTCTTCTGTCCCAACTGGTGCTCCAGCT
GGTGGTGCTGCCGCTGCTGGTGGTGCTGCTCCAGAGGCTGCTGCTGAG
AAGGAAGAGGAGGCCGCTGCCGAGGAGTCTGACGACGACATGGGTTTC GGTTTATTCGATTA SEQ
ID NO 82: >pul36\(P2BF) sequence generated for C. pulcherrima
ACACCTCTCCATCCGCCGCCGATGTCAAGAAGGTCTTGGAGTCCGTTT
CCATCGAGGTTGAGGAGGACAAGGTCGAGAAGTTGCTCGCTGAGGTCG
AGGGCAAGAGCGTCGAGGACTTGATCGCTGAGGGTAACGAGAAGTTGT
CTTCTGTCCCAACTGGTGGCCCAGCCGCCGGTGGTGCCGCTGCCGCTG
CTGGTGGTGACGCCGCTCCTGCCGAGGAGGCCGCTGAGGAGGCCGCCG
AGGAGTCTGACGACGACATGGGTTTCGGTTTATTCGATTA SEQ ID NO 83:
>pul39\(P2BF) sequence generated for C. pulcherrima
CATCCGCCGCCGATGTCAAGRAGGTCTTGGAGTCCGTTTCCATCGAGG
TTGAGGAGGACAAGATCGAGAAGTTGCTCGCTGAGGTCGAGGGCAAGA
GCGTCGAGGACTTGATCGCTGAGGGTAACGAGAAGTTGTCTTCTGTCC
CAACTGGTGGCCCAGCCGCCGGTGGTGCCGCTGCCGCTGCCGGTGGTG
ACGCCGCTCCTGCCGAGGAGGCCGCTGAGGAGGCCGCCGAGGAGTCTG
ACGACGACATGGGTTTCGGTTTATTCGATTA SEQ ID NO 84: >U50\(P2BF)
sequence generated for C. utilis
ACCGCCGACAAGATCACCTCCGTCTTGGAGTCTGTCGGTATTGAGGTT
GAGGAGTCCCAAGTCACCGAGTTGATCTCTGCCCTTGAGGGTAAGTCC
GTTGAGGAGCTCATTGCTGAAGGTAACGAGAAGTTGGCTTCTGTTCCA
ACCGGTGGTGCTGGTGCTGCTCCAGCTGCCGGTGCCGGTGCTGCTGAT
GCTGATGCTCCAGCTGAGGCTGCTGAGGAGGCTGCTGAGGAGGAGTCT
GACGATGACATGGGTTTCGGTTTATTCGATTA Seq ID No 85 P2ForA GAGGAGCGCCT
Seq ID No 86 P2ForB GAGGAGCGCCTC Seq ID No 87 P2ForC GGAGGAGCGCCTC
Seq ID No 88 P2ForD TGAGGAGCGCCTC Seq ID No 89 P2RevA
CCGGAGGGAACGGA Seq ID No 90 P2RevB CCGGAGGGAACGG SeqID No 91 P2FumP
ACTACAGCTCGAAGATTA Seq ID No 92 P2FlavP ACGTTGAATGATTGAGAC Seq ID
No 93 P2NigP TTGCGATTACAAGATGGAA Seq ID No 94 P2TerrP
CTTCGGACTACTGCGATGA Seq ID No 95 CAP2BF (For) ACCTCTCCATCAGCTTCTG
Seq ID No 96 CGP2BF (For) ACCTCTGTCTTATCATCTGTCG Seq ID No 97
CGP2BF (For) AAGAAGGTTATCGAATCTGTTG Seq ID No 98 CTP2BF (For)
TCCGCTTTATTGGAACAAGTTG Seq ID No 99 CPP2BF (For)
TCCTCATTGTTGGAATCCGTTG Seq ID 100 CAP2BR (Rev) TCAGCAATCAATTCTTGC
SEQ ID 101 CKP2BR (Rev) CTCTTCGACGGACTTACC SEQ ID 102 CGP2BR (Rev)
TTCGTCCAAGGACTTACC SEQ ID 103 CTP2BR (Rev) TTCTTGCAAGTCTTTACC SEQ
ID 104 CPP2BR (Rev) CTCGTTGATGTCTTTACC SEQ ID 105 CAP2BP (P)
ACCGCTTTATTGGAATCCGTTG SEQ ID 106 CKP2BP (P)
ATCCGACAAGTTAGACAAGTTAATC SEQ ID 107 CGP2BP (P)
AGAATCAACGAATTGTTGTCTGC SEQ ID 108 CTP2BP (P)
ATCTTCCAAATTAGACTTATTGTTGA SEQ ID 109 CPP2BP (P)
GAAGAATCAAGATTATCTACCTTGTTG
References
[0075] Abramczyk D, Tchorzewski M, Krokowski D, Boguszewska A,
Grankowski N. Overexpression, purification and characterization of
the acidic ribosomal P-proteins from Candida albicans. Biochim
Biophys Acta. 2004 Jun. 11;1672(3):214-23.
[0076] Bailey-Serres J, Vangala S, Szick K, and Lee C H. Acidic
phosphoprotein complex of the 60S ribosomal subunit of maize
seedling roots. Components and changes in response to flooding.
Plant Physiol. 1997 August; 114(4): 1293-1305.
[0077] Newton C H, Shimmin L C, Yee J, Dennis P P. (1990) A family
of genes encode the multiple forms of the Saccharomyces cerevisiae
ribosomal proteins equivalent to the Escherichia coli L12 protein
and a single form of the L10-equivalent ribosomal protein.
JBacteriol. 1990; 172: 579-588
[0078] Tchorzewski M, Krokowski D, Boguszewska A, Liljas A,
Grankowski N. Structural characterization of yeast acidic ribosomal
P proteins forming the P1A-P2B heterocomplex. Biochemistry. 2003
Apr. 1;42(12):3399-408.
[0079] Tchorzewski M, Boguszewska A, Dukowski P, Grankowski N.
Oligomerization properties of the acidic ribosomal P-proteins from
Saccharomyces cerevisiae: effect of P1A protein phosphorylation on
the formation of the P1A-P2B hetero-complex. Biochim Biophys Acta.
2000 Dec 11;1499(1-2):63-73.
[0080] Wool I G, Chan Y L, Gluck A, Suzuki K The primary structure
of rat ribosomal proteins PO, P1 and P2 and a proposal for a
uniform nomenclature for mammalian and yeast ribosomal proteins.
Biochimie 1991; 73: 861-870.
Sequence CWU 1
1
110122DNAArtificial SequencePrimer 1attgttgacc caaggtggta ac
22223DNAArtificial SequencePrimer 2tcgtccaagg acttaccttc caa
23323DNAArtificial SequencePrimer 3tcgtccaagg acttaccttc caa
23422DNAArtificial SequencePrimer 4taatcgaata aaccgaaacc ca
22518DNAArtificial SequencePrimer 5gcgacaagca atttctct
18622DNAArtificial SequencePrimer 6atcatcatca gattcttctt tg
22717DNAArtificial SequencePrimer 7atgaagcacc tcgccgc
17817DNAArtificial SequencePrimer 8agaccgaagc ccatgtc
17929DNAArtificial SequenceProbe based on the P2B/P2 gene for C.
glabrata and A. fumigatus. 9caagaaggtt atcgaatctg ttggtattg
291024DNAArtificial SequenceProbe based on the P2B/P2 gene for C.
glabrata and A. fumigatus. 10cctgccgctg ccggtgccgc tgcc
2411308DNACandida glabrata 11ctattgttga cccaaggtgg taacgaatct
ccagctgctg ctgacatcaa gaaggttatc 60gaatctgttg gtattgaagc tgacgaagcc
agaatcaacg aattgttgtc tgctttggaa 120ggtaagtcct tggacgaatt
gatcgctgaa ggtcaacaaa agttcgcctc tgttccagtt 180ggtggtgctg
ctgctggtgg tgcttccgct gctgctggtg gtgccgctgc cggtgaagcc
240gctgaagaaa aggaagaaga agctgctgaa gaatccgatg acgacatggg
tttcggttta 300ttcgatta 30812305DNACandida glabrata 12ttgttgaccc
aaggtggtaa cgaatctcca gctgctgctg acatcaagaa ggttatcgaa 60tctgttggta
ttgaagctga cgaagccaga atcaacgaat tgttgtctgc tttggaaggt
120aagtccttgg acgaattgat cgctgaaggt caacaaaagt tcgcctctgt
tccagttggt 180ggtgctgctg ctggtggtgc ttccgctgct gctggtggtg
ccgctgccgg tgaagccgct 240gaagaaaagg aagaagaagc tgctgaagaa
tccgatgacg acatgggttt cggtttattc 300gatta 30513308DNACandida
glabrata 13ctattgttga cccaaggtgg taacgaatct ccagctgctg ctgacatcaa
gaaggttatc 60gaatctgttg gtattgaagc tgacgaagcc agaatcaacg aattgttgtc
tgctttggaa 120ggtaagtcct tggacgaatt gatcgctgaa ggtcaacaaa
agttcgcctc tgttccagtt 180ggtggtgctg ctgctggtgg tgcttccgct
gctgctggtg gtgccgctgc cggtgaagcc 240gctgaagaaa aggaagaaga
agctgctgaa gaatccgatg acgacatggg tttcggttta 300ttcgatta
30814308DNACandida glabrata 14ctattgttga cccaaggtgg taacgaatct
ccagctgctg ctgacatcaa gaaggttatc 60gaatctgttg gtattgaagc tgacgaagct
agaatcaacg aattgttgtc tgctttggaa 120ggtaagtcct tggacgaatt
gatcgctgaa ggtcaacaaa agttcgcctc tgttccagtt 180ggtggtgctg
ctgctggtgg tgcttccgct gctgctggtg gtgccgctgc cggtgaagct
240gctgaagaaa aggaagaaga agctgctgaa gaatccgatg acgacatggg
tttcggttta 300ttcgatta 30815296DNACandida krusei 15ttactcttag
tcaatgctgg taaaaccgca ccatctgctg cagatgttac ctctgtctta 60tcatctgtcg
gtatcgaagt tgaatccgac aagttagaca agttaatctc cgaattagaa
120ggtaagtccg tcgaagagtt gattgctgaa ggtactgaaa agatggcttc
tgctccaggt 180gcagcagctg ctccagcttc tggtgcaggt gcttccaccg
aatctgctgc agcagaagaa 240gttgaagaag aaaaggaaga atccgatgat
gacatgggtt tcggtttatt cgatta 29616265DNACandida krusei 16catctgctgc
agatgttacc tctgtcttat catctgtcgg tatcgaagtt gaatccgaca 60agttagacaa
gttaatctcc gaattagaag gtaagtccgt cgaagagttg attgctgaag
120gtactgaaaa gatggcttct gctccaggtg cagcagctgc tccagcttct
ggtgcaggtg 180cttccaccga atctgctgca gcagaagaag ttgaagaaga
aaaggaagaa tccgatgatg 240acatgggttt cggtttattc gatta
26517265DNACandida krusei 17catctgctgc agatgttacc tctgtcttat
catctgtcgg tatcgaagtt gaatccgaca 60agttagacaa gttaatctcc gaattagaag
gtaagtccgt cgaagagttg attgctgaag 120gtactgaaaa gatggcttct
gctccaggtg cagcagctgc tccagcttct ggtgcaggtg 180cttccaccga
atctgctgca gcagaagaag ttgaagaaga aaaggaagaa tccgatgatg
240acatgggttt cggtttattc gatta 26518296DNACandida krusei
18ttactcttag tacatgctgg taaaaccgca ccatctgctg cagatgttac ctctgtctta
60tcatctgtcg gtatcgaagt tgaatccgac aagttagaca agttaatctc cgaattagaa
120ggtaagtccg tcgaagagtt gattgctgaa ggtactgaaa agatggcttc
tgctccaggt 180gcagcagctg ctccagcttc tggtgcaggt gcttccaccg
aatctgctgc agcagaagaa 240gttgaagaag aaaaggaaga atccgatgat
gacatgggtt tcggtttatt cgatta 29619331DNACandida albicans
19ttattgttag ttcaaggtgg taacacctct ccatcagctt ctgatatcac cgctttattg
60gaatccgttg gtgttgaagc cgaagaatcc agattacaag ctttattgaa agatttggaa
120ggtaaagact tgcaagaatt gattgctgaa ggtaacacca aattagcttc
tgtcccatcc 180ggtggtgctg ctgctggtgg tgcttctgcc tctgctggtg
ccgctgctgg tggtgctgct 240gaagctgaag aagaaaaaga agaagaagcc
aaagaagaat ctgatgatga tatgggtttc 300ggtttattcg attagagaaa
ttgcttgtcg c 33120331DNACandida albicans 20ttattgttag ttcaaggtgg
taacacctct ccatcagctt ctgatatcac cgctttattg 60gaatccgttg gtgttgaagc
cgaagaatcc agattacaag ctttattgaa agatttggaa 120ggtaaagact
tgcaagaatt gattgctgaa ggtaacacca aattagcttc tgtcccatcc
180ggtggtgctg ctgctggtgg tgcttctgcc tctgctggtg ccgctgctgg
tggtgctgct 240gaagctgaag aagaaaaaga agaagaagcc aaagaagaat
ctgatgatga tatgggtttc 300ggtttattcg attagagaaa ttgcttgtcg c
33121331DNACandida albicans 21ttattgttag ttcaaggtgg taacacctct
ccatcagctt ctgatatcac cgctttattg 60gaatccgttg gtgttgaagc cgaagaatcc
agattacaag ctttattgaa agatttggaa 120ggtaaagact tgcaagaatt
gattgctgaa ggtaacacca aattagcttc tgtcccatcc 180ggtggtgctg
ctgctggtgg tgcttctgcc tctgctggtg ccgctgctgg tggtgctgct
240gaagctgaag aagaaaaaga agaagaagcc aaagaagaat ctgatgatga
tatgggtttc 300ggtttattcg attagagaaa ttgcttgtcg c 33122331DNACandida
albicans 22ttattgttag ttcaaggtgg taacacctct ccatcagctt ctgatatcac
cgctttattg 60gaatccgttg gtgttgaagc ygaagaatcc agattacaag ctttattgaa
agatttggaa 120ggtaaagact tgcaagaatt gattgctgaa ggtaacacca
aattagcttc tgtcccatcc 180ggtggtgctg ctgctggtgg tgcttctgcc
tctrctggtg ccgctgctgg yggtgctgcy 240gaagctgaag aagaaaaaga
agaagaagcc aaagaagaat ctgatgatga tatgggtttc 300ggtttattcg
attagagaaa ttgcttgtcg c 33123331DNACandida dubliniensis
23ttgttgttag ttcaaggtgg taacgccact ccatcagctt ctgatatcag cgctgtcttg
60gaaactgttg gtgttgaagc cgaagaatcc agattacaag ctttattgaa agatttggaa
120ggtaaagatt tgcaagaatt gattgctgaa ggtaacacca aattagcttc
tgtcccaacc 180ggtggtgctg ctgctggtgg tgcttccggt tctgctggtg
ccgcttctgg tgccgctgct 240gaagctgaag aagaaaaaga agaagaagct
aaagaagaat ctgatgatga tatgggtttc 300ggtttattcg attagagaaa
ttgcttgtcg c 33124292DNACandida dubliniensis 24ttgttgttag
ttcaaggtgg taacgccact ccatcagctt ctgatatcag cgctgtcttg 60gaaactgttg
gtgttgaagc cgaagaatcc agattacaag ctttattgaa agatttggaa
120ggtaaagatt tgcaagaatt gattgctgaa ggtaacacca aattagcttc
tgtcccaacc 180ggtggtgctg ctgctggtgg tgcttccggt tctgctggtg
ccgcttctgg tgccgctgct 240gaagctgaag aagaaaaaga agaagaagcc
aaagaagaat ctgatgatga ta 29225338DNACandida parapsilosis
25atgaaatact tagctgctta cttattattg gtccaaggtg gtaacgcctc cccatctgct
60tcagacatct cctcattgtt ggaatccgtt ggtgttgaag ttgaagaatc aagattatct
120accttgttga aagacttgga aggtaaagac atcaacgagt tgattgctga
aggtaacacc 180aaattggcct cagttccatc tggtggtgct gctgttgctt
ccggttctgg tgcttctggt 240gccgctgctg gtggtgctgc tgaagaagct
aaggaagaag ccaaggaaga agaaaaggaa 300gaatctgatg atgacatggg
tttcggttta ttcgatta 33826317DNACandida parapsilosis 26ttattattgg
tccaaggtgg taacgcctcc ccatctgctt cagacatctc ctcattgttg 60gaatccgttg
gtgttgaagt tgaagaatca agattatcta ccttgttgaa agacttggaa
120ggtaaagaca tcaacgagtt gattgctgaa ggtaacacca aattggcctc
agttccatct 180ggtggtgctg ctgttgcttc cggttctggt gcttctggtg
ccgctgctgg tggtgctgct 240gaagaagcta aggaagaagc caaggaagaa
gaaaaggaag aatctgatga tgacatgggt 300ttcggtttat tcgatta
31727255DNACandida parapsilosis 27ggtggtaacg cctccccatc cgcttcagac
atctcatcct tgttggaatc cgttggtgtt 60gaagttgaag aatcaagatt gtccctcttg
ttgaaagact tggaaggtaa agacatcaac 120gaattgattg ctgaaggtaa
caccaagttg gcttcagttc caactggtgg tgctgctgtt 180gcttctggtt
ctggtgcttc aggtgccgct gctggtggtg ctgctgaaga agccaaagaa
240gaatctgatg atgat 25528273DNACandida tropicalis 28ggtggtaacg
cttccccatc tgcttctgac atctccgctt tattggaaca agttggtgct 60gaagttgaat
cttccaaatt agacttattg ttgaaagaat tggaaggtaa agacttgcaa
120gaattgattg ccgaaggtaa cactaaattc gcctctgtcc catccggtgg
tgctgctgct 180gcttcytctg gttccgctgc cgctgctggt ggtgccgctg
ccgaagctga agaagaaaaa 240gaagaagaag ccaaagaaga atctgatgat gat
27329273DNACandida tropicalis 29ggtggtaacg cttccccatc tgcttctgac
atctccgctt tattggaaca agttggtgct 60gaagttgaat cttccaaatt agacttattg
ttgaaagaat tggaaggtaa agacttgcaa 120gaattgattg ccgaaggtaa
cactaaattc gcctctgtcc catccggtgg tgctgctgct 180gcttcctctg
gttccgctgc cgctgctggt ggtgccgctg ccgaagctga agaagaaaaa
240gaagaagaag ccaaagaaga atctgatgat gat 27330288DNACandida
tropicalis 30ttattattag tccaaggtgg taacrcttcc ccatctgctt ctgacatctc
cgctttattg 60gaacaagttg gtgctgaagt tgaatcttcc aaattagact tattgttgaa
agaattggaa 120ggtaaagact tgcaagaatt gattgccgaa ggtaacacta
aattcgcctc tgtcccatcc 180ggtggtgctg cygctgcttc ctctggttcc
gctgccgctg ctggtggtgc cgctgccgaa 240gctgaagaag aaaaagaaga
agaagccaaa gaagaatctg atgatgat 28831288DNACandida tropicalis
31ttattattag tccaaggtgg taacgcttcc ccatctgctt ctgacatctc cgctttattg
60gaacaagttg gtgctgaagt tgaatcttcc aaattagact tattgttgaa agaattggaa
120ggtaaagact tgcaagaatt gattgccgaa ggtaacacta aattcgcctc
tgtcccatcc 180ggtggtgctg ctgctgcttc ctctggttcc gctgccgctg
ctggtggtgc cgctgccgaa 240gctgaagaag aaaaagaaga agaagccaaa
gaagaatctg atgatgat 28832288DNACandida tropicalis 32ttattattag
tccaaggtgg taacgcttcc ccatctgctt ctgacatctc cgctttattg 60gaacaagttg
gtgctgaagt tgaatcttcc aaattagact tattgttgaa agaattggaa
120ggtaaagact tgcaagaatt gattgccgaa ggtaacacta aattcgcctc
tgtcccatcc 180ggtggtgctg ctgctgcttc ttctggttcc gctgccgctg
ctggtggtgc cgctgccgaa 240gctgaagaag aaaaagaaga agaagccaaa
gaagaatctg atgatgat 28833302DNACandida guilliermondii 33ttrttgttgg
tkracgccgg taacacctcc ccatctgctg ctgacatcaa ggctgtcttg 60gagtcggttt
ccatagaagt tgacgacgag aaggtgtcca agttgttgag cgaagttgag
120ggaaagaatg ctgaagaatt gatcgctgaa ggtaacgaaa aattgtcttc
tgttccaact 180ggtggaccag ctgctgcttc ctctggatct gctgccgctg
ccgatgctcc tgctgccgaa 240gaggccgctg aggaggccgc tgaggagtct
gacgacgaca tgggtttcgg tttattcgat 300ta 30234302DNACandida
guilliermondii 34ttrttgttgg tkracgccgg taacacctcc ccatctgctg
ctgacatcaa ggctgtcttg 60gagtcggttt ccatagaagt tgacgacgag aaggtgtcca
agttgttgag cgaagttgag 120ggaaagaatg ctgaagaatt gatcgctgaa
ggtaacgaaa aattgtcttc tgttccaact 180ggtggaccag ctgctgcttc
ctctggatct gctgccgctg ccgatgctcc tgctgccgaa 240gaggccgctg
aggaggccgc tgaggagtct gacgacgaca tgggtttcgg tttattcgat 300ta
30235302DNACandida lusitanie 35ttattgttgg tcaacgctgg taacaccgcc
ccatctgctg ctgacgtcaa gaaggtcttg 60gaatccgtct ctattgaggt tgaggacgac
aaggttgaga agttgttggc tgaagttgaa 120ggcaagaacg tcgaagagtt
gattgccgag ggtaacgaga agttgtcttc tgttccatct 180ggtgctccag
ctgctgctgg tgccgctgct gcttctggtt ctactgaggc tgctgctgaa
240gagccacaag aagaagagaa ggaggagtct gacgacgaca tgggtttcgg
tttattcgat 300ta 30236380DNAAspergillus fumigatus 36ttacctcctc
ctcgcccttg ctggcaacac ctccccgtcc tctgaggatg tcaaggccgt 60cctctcttcc
gttggcattg atgccgatga ggagcgcctg aacaagctca ttgctgagct
120cgagggcaag gacctccagg aggttagtaa ctacagctcg aagattacag
actgggaatt 180ttggactggc gctgacatcg aactctacaa cagctcattg
ccgagggttc caccaagctc 240gcttccgttc cctccggtgg tgctgccgcc
gctgctcctg ccgctgccgg tgccgctgcc 300ggtggtgctg ctgctcctgc
cgctgaggag aagaaggagg aggagaagga ggagtccgac 360gaggacatgg
gcttcggtct 38037377DNAAspergillus fumigatus 37cctcctcctc gcccttgctg
gcaacacctc cccgtcctct gaggatgtca aggccgtcct 60ctcttccgtt ggcattgatg
ccgatgagga gcgcctgaac aagctcattg ctgagctcga 120gggcaaggac
ctccaggagg ttagtaacta cagctcgaag attacagact gggaattttg
180gactggcgct gacatcgaac tctacaacag ctcatcgccg agggttccac
caagctcgct 240tccgttccct ccggtggtgc tgccgccgct gctcctgccg
ctgccggtgc cgctgccggt 300ggtgctgctg ctcctgccgc tgaggagaag
aaggaggagg agaaggagga gtccgacgag 360gacatgggct tcggtct
37738357DNAAspergillus fumigatus 38gcaacacctc cccgtcctct gaggatgtca
aggccgtcct ctcttccgtt ggcattgatg 60ccgatgagga gcgcctgaac aagctcattg
ctgagctcga gggcaaggac ctccaggagg 120ttagtaacta cagctcgaag
attacagact gggaattttg gactggcgct gacatcgaac 180tctacaacag
ctcattgccg agggttccac caagctcgct tccgttccct ccggtggtgc
240tgccgccgct gctcctgccg ctgccggtgc cgctgccggt ggtgctgctg
ctcctgccgc 300tgaggagaag aaggaggagg agaaggagga gtccgacgag
gacatgggct tcggtct 35739380DNAAspergillus fumigatus 39ttacctcctc
ctcgcccttg ctggcaacac ctccccgtcc tctgaggatg tcaaggccgt 60cctctcttcc
gttggcattg atgccgatga ggagcgcctg aacaagctca ttgctgagct
120cgagggcaag gacctccagg aggttagtaa ctacagctcg aagattacag
actgggaatt 180ttggactggc gctgacatcg aactctacaa cagctcattg
ccgagggttc caccaagctc 240gcttccgttc cctccggtgg tgctgccgcc
gctgctcctg ccgctgccgg tgccgctgcc 300ggtggtgctg ctgctcctgc
cgctgaggag aagaaggagg aggagaagga ggagtccgac 360gaggacatgg
gcttcggtct 38040380DNANeosartorya fischeri 40ttacctcctc ctcgcccttg
ctggcaacac ctccccctcc gctgaggatg tcaaggccgt 60cctctcttcc gtcggcattg
acgccgatga ggagcgcctg aacaagctca ttgctgagct 120cgagggcaag
gacctccagg aggttagtac acacggcttg aatattaccg actgagaatt
180ttggaccggc gctgacatcg atttctacaa cagctgatcg ctgagggttc
cgccaagctc 240gcttccgttc cctccggtgg tgccggtggt gccgctgctc
ctgccgctgg cggtgccgct 300gccggtggtg ctgctgccgc tcccgccgaa
gagaaggagg aggagaagga ggagtccgac 360gaggacatgg gcttcggtct
38041365DNAAspergillus nidulans 41ctacctcctc ctcgcccttg ctggcaacga
gtctccctcc gcctccgaca tcaaggaggt 60tctctcttcc gtcggtgttg acgccgacga
cgagcgcctc gagaagctca ttgctgagct 120ccagggcaag gacatcaacg
aggttcgtta ttgcatatag agttggaaga cgcggactgc 180gggctaacga
taatctttaa acagctgatc gctgagggta ccaccaagct tgcctccgtt
240ccctccggcg gtgctggtgg tgctgcccct gctgccgctg ccggtggtgc
tgctgctgcc 300gaggcccccg ctgctgagaa ggaggaggag aaggaggagt
ccgatgagga catgggcttc 360ggtct 36542349DNAAspergillus nidulans
42ccttgctggc aacgagtctc cctccgcctc cgacatcaag gaggttctct cttccgtcgg
60tgttgacgcc gacgacgagc gcctcgagaa gctcattgct gagctccagg gcaaggacat
120caacgaggtt cgttattgca tagggttgga agacgcggac agcgggctaa
cgataatctt 180ctgaacagct gatcgctgag ggtaccacca agcttgcctc
cgttcccacc ggcggtgctg 240gtgctgctgc ccctgctgcc gctgccggtg
gtgctgccgc tgccgaggct cccgctgctg 300agaaggagga ggagaaggag
gagtccgatg aggacatggg cttcggtct 34943376DNAAspergillus clavatus
43ttacctcctc ctcgcccttg gtggcaacgc cagcccctcc gctgctgatg ttaaggaggt
60tctctcttct gtcggcattg atgctgatga ggagcgcctc aacaagctca ttgctgagct
120cgagggcaag gaccttcagg aggttagttt tgcgctggtc tacgagagga
agattgtgac 180aagatgctaa cggaaaattt cttcaacagc tgattgctga
gggttccacc aagctcgctt 240ccattccctc cggcggtgct ggtggtgctg
cccccgccgc tggcggtgct gccgccggtg 300gtgctgctga ggccgctccc
gctgaggaga aggaggagga gaaggaggag tccgacgacg 360acatgggctt cggtct
37644385DNAAspergillus niger 44ctaccttctg ttggcccttg ctggcaacaa
caccccctcc gctgaggaca tcaagtccgt 60cctctccgcc gtcggcattg acgctgagga
ggagcgcctc cagaagctcc ttgctgagct 120tgagggcaag gacctccagg
aggtcagtta acgcccttaa aatctaccaa gaaattttgc 180gattacaaga
tggaatactg acaatgggtt ttctacaaca gctcatctcc gagggtaccc
240agaagctcgc ttccgttccc tccggtggtg ccggtgctgc tgccgctgcc
cccgctgccg 300gtggcgccgc tgctgctgag gctcccgctg aggagaagaa
ggaggaggct gctgaggagt 360ccgatgagga catgggcttc ggtct
38545385DNAAspergillus niger 45ctaccttctg ttggcccttg ctggcaacaa
caccccctcc gctgaggaca tcaagtccgt 60cctctccgcc gtcggcattg acgctgagga
ggagcgcctc cagaagctcc ttgctgagct 120tgagggcaag gacctccagg
aggtcagtta acgcccttaa aatctaccaa gaaattttgc 180gattacaaga
tggaatactg acaatggttt ttctacaaca gctcatctcc gagggtaccc
240agaagctcgc ttccgttccc tccggtggtg ccggtgctgc tgccgctgcc
cccgctgccg 300gtggcgccgc tgctgctgag gctcccgctg aggagaagaa
ggaggaggct gctgaggagt 360ccgatgagga catgggcttc ggtct
38546366DNAAspergillus terreus 46ttccttctcc tcggccttgc cggcaacacc
tccccctctg ctgaggacat caaggctgtc 60ctctcctccg tcggcattga cgctgatgag
gagcgcctcg gccagctcct gaaggagctc 120gagggcaagg acatccagga
ggttagtgat caccataact tcggactact gcgatgagaa 180cgcgccatac
taacggatta tacagctcat cgctcagggc tctgagaagc tcgcttccgt
240tccctctggc ggtgctgccg ctgctgctgc tcccgccgct gccgctggcg
gtgacgctgc 300tgcccccgct gagaagaagg aggaggagaa ggaggaggag
tccgacgagg acatgggctt 360cggtct 36647363DNAAspergillus flavus
47ttacctcctc ctcgccctcg ctggcaactc caccccctcc gttgaggaca tcaagagcgt
60tctctcttcc gtcggtattg atgccgatga ggagcgcctc cagaaggtca
tctccgagct
120cgagggcaag gacctccagc aggttcgtaa tacgttgaat gattgagaca
tagggcgcct 180gctgaccatc tactcactac agctgatcac tgagggtagc
gagaagctcg ctaccgttcc 240ctccggtggt gctggtgccg ctgcccctgc
tgctggcggt gccgctgccg gtggtgacgc 300ccccgccgct gaggagaagg
aggaagagaa ggaggagtcc gatgaggaca tgggcttcgg 360tct
36348351DNAAspergillus versicolor 48tcgcccttgc tggcaacgag
agcccctctg cttccgacat taaggaggtt ctgtcctccg 60tcggtgttga cgctgacaac
gagcgcctcg agaagctcat cgctgagctc cagggcaagg 120acatcaacga
ggttcgtttt gacagatgca tttgaaatac ttggccagca gactaatgaa
180acctcttctg cagttgatcg ctgagggtac caccaagctc gcttccgttc
cctctggcgg 240tggtggtggt gctgccgccc ccgctgctgg tggcgctgcc
gccgctgagg cccctgctgc 300tgagaaggag gaggagaagg aggagtccga
cgaggacatg ggcttcggtc t 35149348DNAAspergillus versicolor
49cccttgctgg caacgagagc ccctctgctt ccgacattaa ggaggttctg tcctccgtcg
60gtgttgacgc tgacgacgag cgcctcgaga agctcatcgc tgagctccag ggcaaggaca
120tcaacgaggt tcgttttgac agatgcgttt gaaatacttg gccagcagac
taatgaaacc 180tcttctgcag ttgatcgctg agggtaccac caagctcgct
tccgttccct ctggcggtgg 240tggtggtgct gccgcccccg ctgctggtgg
cgctgccgcc gctgaggccc ctgctgctga 300gaaggaggag gagaaggagg
agtccgacga ggacatgggc ttcggtct 34850481DNACandida albicans
50ggaaaaaatg aaatacttag ctgcttactt attgttagtt caaggtggta acacctctcc
60atcagcttct gatatcaccg ctttattgga atccgttggt gttgaagccg aagaatccag
120attacaagct ttattgaaag atttggaagg taaagacttg caagaattga
ttgctgaagg 180taacaccaaa ttagcttctg tcccatccgg tggtgctgct
gctggtggtg cttctgcctc 240tactggtgcc gctgctggtg gtgctgccga
agctgaagaa gaaaaagaag aagaagccaa 300agaagaatct gatgatgata
tgggtttcgg tttattcgat tagagaaatt gcttgtcgcc 360tttgctggtt
tgagagaagt atatttccat tattttgcat tatatatata tatatgtatt
420ataactaatc taataaaaaa aatatgaaaa caaaaatggc ttctatatgg
cactgtttgc 480a 48151330DNACandida glabrata 51atgaagtact tggccgctta
cctattgttg acccaaggtg gtaacgaatc tccagctgct 60gctgacatca agaaggttat
cgaatctgtt ggtattgaag ctgacgaagc tagaatcaac 120gaattgttgt
ctgctttgga aggtaagtcc ttggacgaat tgatcgctga aggtcaacaa
180aagttcgcct ctgttccagt tggtggtgct gctgctggtg gtgcttccgc
tgctgctggt 240ggtgccgctg ccggtgaagc tgctgaagaa aaggaagaag
aagctgctga agaatccgat 300gacgacatgg gtttcggttt gttcgactaa
33052336DNACandida albicans 52atgaaatact tagctgctta cttattgtta
gttcaaggtg gtaacacctc tccatcagct 60tctgatatca ccgctttatt ggaatccgtt
ggtgttgaag ccgaagaatc cagattacaa 120gctttattga aagatttgga
aggtaaagac ttgcaagaat tgattgctga aggtaacacc 180aaattagctt
ctgtcccatc cggtggtgct gctgctggtg gtgcttctgc ctctgctggt
240gccgctgctg gtggtgctgc tgaagctgaa gaagaaaaag aagaagaagc
caaagaagaa 300tctgatgatg atatgggttt cggtttattc gattag
33653336DNACandida albicans 53atgaaatact tagctgctta cttattgtta
gttcaaggtg gtaacacctc tccatcagct 60tctgatatca ccgctttatt ggaatccgtt
ggtgttgaag ccgaagaatc cagattacaa 120gctttattga aagatttgga
aggtaaagac ttgcaagaat tgattgctga aggtaacacc 180aaattagctt
ctgtcccatc cggtggtgct gctgctggtg gtgcttctgc ctctgctggt
240gccgctgctg gtggtgctgc tgaagctgaa gaagaaaaag aagaagaagc
caaagaagaa 300tctgatgatg atatgggttt cggtttattc gattag
33654336DNAAspergillus fumigatus 54atgaagcacc tcgccgctta cctcctcctc
gcccttgctg gcaacacctc cccgtcctct 60gaggatgtca aggccgtcct ctcttccgtt
ggcattgatg ccgatgagga gcgcctgaac 120aagctcattg ctgagctcga
gggcaaggac ctccaggagc tcatcgccga gggttccacc 180aagctcgctt
ccgttccctc cggtggtgct gccgccgctg ctcctgccgc tgccggtgcc
240gctgccggtg gtgctgctgc tcctgccgct gaggagaaga aggaggagga
gaaggaggag 300tccgacgagg acatgggctt cggtcttttc gactaa
33655336DNANeosartorya fischeri 55atgaagcacc tcgccgctta cctcctcctc
gcccttgctg gcaacacctc cccgtccgct 60gaggatgtca aggccgtcct ctcttccgtt
ggcattgacg ccgatgagga gcgcctgaac 120aagctcattg ctgagctcga
gggcaaggac ctccaggagc tcatcgccga gggttccacc 180aagctcgctt
ccgttccctc cggtggtgct gccgccgctg ctcctgccgc tggcggtgcc
240gctgccggtg gtgctgctgc tcctgccgct gaggagaaga aggaggagga
gaaggaggag 300tccgacgagg acatgggctt cggtctcttc gactaa
33656336DNAAspergillus clavatus 56atgaagcacc tcgccgctta cctcctcctc
gcccttggtg gcaacgccag cccctccgct 60gctgatgtta aggaggttct ctcttctgtc
ggcattgatg ctgatgagga gcgcctcaac 120aagctcattg ctgagctcga
gggcaaggac cttcaggagc tgattgctga gggttccacc 180aagctcgctt
ccattccctc cggcggtgct ggtggtgctg cccccgccgc tggcggtgct
240gccgccggtg gtgctgctga ggccgctccc gctgaggaga aggaggagga
gaaggaggag 300tccgacgacg acatgggctt cggtctcttc gactaa
33657333DNAAspergillus terreus 57atgaagcacc tcgccgcttt ccttctcctc
ggccttgccg gcaacacctc cccctctgct 60gaggacatca aggctgtcct ctcctccgtc
ggcattgacg ctgatgagga gcgcctcggc 120cagctcctga aggagctcga
gggcaaggac atccaggagc tcatcgctca gggctctgag 180aagctcgcct
ccgttccctc tggcggtgct gccgctggtg ctgctgctcc cgccgctgcc
240gctggcggtg acgctgctgc ccccgctgag aagaaggagg aggagaagga
ggaggagtcc 300gacgaggaca tgggcttcgg tctcttcgac taa
33358333DNAAspergillus niger 58atgaagtacc tcgccgccta ccttctgttg
gcccttgctg gcaacaacac cccctccgct 60gaggacatca agtccgtcct ctccgccgtc
ggcattgacg ctgaggagga gcgcctccag 120aagctccttg ctgagcttga
gggcaaggac ctccaggagc tcatctccga gggtacccag 180aagctcgctt
ccgttccctc cggtggtgcc ggtgctgctg ccgctgcccc cgctgccggt
240ggcgccgctg ctgctgaggc tcccgctgag gagaagaagg aggaggctgc
tgaggagtcc 300gatgaggaca tgggcttcgg tctcttcgac taa
33359330DNAAspergillus nidulans 59atgaagcacc tcgcagccta cctcctcctc
gcccttgctg gcaacgagtc tccctccgcc 60tccgacatca aggaggttct ctcttccgtc
ggtgttgacg ccgacgacga gcgcctcgag 120aagctcattg ctgagctcca
gggcaaggac atcaacgagc tgatcgctga gggtaccacc 180aagcttgcct
ccgttccctc cggcggtgct ggtggtgctg cccctgctgc cgctgccggt
240ggtgctgctg ctgccgaggc ccccgctgct gagaaggagg aggagaagga
ggagtccgat 300gaggacatgg gcttcggtct cttcgactaa 33060476DNACandida
albicans 60gaattcgatt attgcattct gatattccct gctttaaatg catttggaaa
tatttcgtat 60atcatgagat ataataacat taatagcatt ttcatgttac taacaagaat
atagtgaaat 120acttagctgc ttacttatta ttagttaacg ccggtaacgc
caccccatct gctgccgatg 180tcaaagctgt tttgtcagct gctgatattg
aagtcgaaga agaaaaagtt gaaaaattga 240tcagcgaatc ggacggtaag
aacgtcgaag aattgattgc tgaaggtaac gaaaaattat 300catcagtccc
atctggtgct ccagctgctg ctgctggtgg tgcttctgct gccgccggtg
360gtgaagccac tgaagaagct gctgaagaag aagctgctga agaatctgat
gacgatatga 420gtttcggttt attcgattaa acgagtcaac aggcatctca
agatcacagc ataagg 47661352DNAAspergillus flavus 61tcgccctcgc
tggcaactcc accccctccg ttgaggacat caagagcgtt ctctcttccg 60tcggtattga
tgccgatgag gagcgcctcc agaaggtcat ctccgagctc gagggcaagg
120acctccagca ggttcgtaat acgttgaatg attgagacat agggcgcctg
ctgaccatct 180actcactaca gctgatcact gagggtagcg agaagctcgc
taccgttccc tccggtggtg 240ctggtgccgc tgcccctgct gctggcggtg
ccgctgccgg tggtgacgcc cccgccgctg 300aggagaagga ggaagagaag
gaggagtccg atgaggacat gggcttcggt ct 35262386DNAAspergillus niger
62ctaccttctg ttggcccttg ctggcaacaa caccccctcc gctgaggaca tcaagtccgt
60cctctccgcc gtcggcattg acgctgagga ggagcgcctc cagaagctcc ttgctgagct
120tgagggcaag gacctccagg aggtcagtta acgcccttaa aaatctacca
agaaattttg 180cgattacaag atggaatact gacaatggtt tttctacaac
agctcatctc cgagggtacc 240cagaagctcg cttccgttcc ctccggtggt
gccggtgctg ctgccgctgc ccccgctgcc 300ggtggcgccg ctgctgctga
ggctcccgct gaggagaaga aggaggaggc tgctgaggag 360tccgatgagg
acatgggctt cggtct 38663370DNAAspergillus terreus 63tttccttctc
ctcggccttg ccggcaacac ctccccctct gctgaggaca tcaaggctgt 60cctctcctcc
gtcggcattg acgctgatga ggagcgcctc ggccagctcc tgaaggagct
120cgagggcaag gacatccagg aggttagtga tcaccataac ttcggactac
tgcgatgaaa 180acgcgccata ctaacggatt atacagctca tcgctcaggg
ctctgagaag ctcgcctccg 240ttccctctgg cggtgctgcc gctggtgctg
ctgctcccgc cgctgccgct ggcggtgacg 300ctgctgcccc cgctgagaag
aaggaggagg agaaggagga ggagtccgac gaggacatgg 360gcttcggtct
37064370DNAAspergillus terreus 64tttccttctc ctcggccttg ccggcaacac
ctccccctct gctgaggaca tcaaggctgt 60cctctcctcc gtcggcattg acgctgatga
ggagcgcctc ggccagctcc tgaaggagct 120cgagggcaag gacatccagg
aggttagtga tcaccataac ttcggactac tgcgatgaaa 180acgcgccata
ctaacggatt atacaggtca tcgctcaggg ctctgagaag ctcgcctccg
240ttccctctgg cggtgctgcc gctggtgctg ctgctcccgc cgctgccgct
ggcggtgacg 300ctgctgcccc cgctgagaag aaggaggagg agaaggagga
ggagtccgac gaggacatgg 360gcttcggtct 37065370DNAAspergillus terreus
65tttccttctc ctcggccttg ccggcaacac ctccccctct gctgaggaca tcaaggctgt
60cctctcctcc gtcggcattg acgctgatga ggagcgcctc ggccagctcc tgaaggagct
120cgagggcaag gacatccagg aggttagtga tcaccataac ttcggactac
tgcgatgaaa 180acgcgccata ctaacggatt atacagctca tcgctcaggg
ctctgagaag ctcgcctccg 240ttccctctgg cggtgctgcc gctggtgctg
ctgctcccgc cgctgccgct ggcggtgacg 300ctgctgcccc cgctgagaag
aaggaggagg agaaggagga ggagtccgac gaggacatgg 360gcttcggtct
37066370DNAAspergillus terreus 66tttccttctc ctcggccttg ccggcaacac
ctccccctct gctgaggaca tcaaggctgt 60cctctcctcc gtcggcattg acgctgatga
ggagcgcctc ggccagctcc tgaaggagct 120cgagggcaag gacatccagg
aggttagtga tcaccataac ttcggactac tgcgatgaaa 180acgcgccata
ctaacggatt atacagctca tcgctcaggg ctctgagaag ctcgcctccg
240ttccctctgg cggtgctgcc gctggtgctg ctgctcccgc cgctgccgct
ggcggtgacg 300ctgctgcccc cgctgagaag aaggaggagg agaaggagga
ggagtccgac gaggacatgg 360gcttcggtct 37067356DNAAspergillus candidus
67tgctcctcgg cctcgcmggc aacgagactc cctccgctgc cgacatcaag ggcgttctgt
60ccgccgtcgg cattgacgcc gatgaggacc gtctctccaa gctcctctcc gagcttgagg
120gcaaggacat caacgaggtt cgtatctcac aggaatcgca cacgtaacag
agtcaacaaa 180tactaatccc ccgtgcagct gatcgcccag ggctccgaga
agcttgcttc cgttccctcc 240ggtggtgccg ctggtggtgc cgctgccgcc
cctgccgccg ccgctggtgg tgacgctccc 300gcccaggaga aggaggagga
gaaggaggag tccgatgagg acatgggctt cggtct 35668352DNAAspergillus
candidus 68tgctcctcgg cctcgccggc aacgagtctc cctccgctgc cgacatcaag
ggcgttctgt 60ccgccgtcgg cattgacgcc gatgaggagc gtctctccaa gctcctctcc
gagcttgagg 120gcaaggacat caacgaggtt cgtatcctgg aatcgcaccg
agacagagtc aacaaatact 180aatcccccgt gcagctgatc gcccagggta
ccgagaagct tgcttccgtt ccctccggtg 240gcgccggcgc tgctgctgcc
gcccctgccg ccgctgctgg tggtgaggcc gccgctgagg 300agaagaagga
ggaggagaag gaggagtccg atgaggacat gggcttcggt ct
35269336DNAAspergillus candidus 69ggcaaccgag tctccctccg ctgccgacat
caagggcgtt ctgtccgccg tcggcattga 60cgccgatgag gagcgtctct ccaagctcct
ctccgagctt gagggcaagg acatcaacga 120ggttcgtatc ctggaatcgc
accgagacag agtcaacaaa tactaatccc ccgtgcagct 180gatcgcccag
ggtaccgaga agcttgcttc cgttccctcc ggtggcgccg gcgctgctgc
240tgccgcccct gccgccgctg ctggtggtga ggccgccgct gaggagaaga
aggaggagga 300gaaggaggag tccgatgagg acatgggctt cggtct
33670376DNAAspergillus clavatus 70ttacctcctc ctcgcccttg gtggcaacgc
cagcccctcc gctgctgatg ttaaggaggt 60tctctcttcc gtcggcattg atgctgatga
ggagcgcctc aacaagctca ttgctgagct 120cgagggcaag gaccttcagg
aggttagttt tacgctggtc tacgagagga agattgtgac 180aagatgctaa
cggaaaattt cttcaacagc tgattgctga gggttccacc aagctcgctt
240ccattccctc cggcggtgct ggtggtgctg cccccgccgc tggcggtgct
gccgccggtg 300gtgctgctga ggccgctccc gctgaggaga aggaggagga
gaaggaggag tccgacgacg 360acatgggctt cggtct 37671372DNAAspergillus
clavatus 71ttacctcctc ctcgcccttg gtggcaacgc cagcccctcc gctgctgatg
ttaaggaggt 60tctctcttcc gttggcattg atgctgatga ggagcgcctc aataagctca
ttgctgagct 120tgagggcaag gaccttcagg aggttagttt acgccaatcc
gcgatatgaa aattgcgaca 180tgatgctaac aggagaattt ttcaacagct
gattgctgag ggttccacca agctcgcttc 240cattccttcc ggcggtgctg
ccggtgctgc ccctgccgct ggcgctgctg ccggtggtgc 300cgctgaggcc
gctcccgctg aggagaagga ggaggagaag gaggagtccg acgacgacat
360gggcttcggt ct 37272431DNAAspergillus glaucus 72acacggtgga
ctggctggcc ggaagagccg ttgctggtgt aagttttggt aggaccgttc 60agctccrgak
gtctttgggg acaatggcga cgagggctgt cgatcgttag tgtacattcc
120aaggaaggta aaagaaaaag aatgcgacat acagtaagtt gagccactct
gacgctggca 180gtcatcgcag tggtcataag ctgtagcgag cggctgctcg
atgttgaccg agtaggtgac 240ggctttgcag agacagtgac cggaaatagg
cattttggac gagaggcaga atataattgg 300actatagact gacaatgatg
atgacgatac tgaggacctt tgggaggaat gaaacagata 360tttataccct
ctccgacctc gtgtagtcca tctgtctttc gctgtctctc ggtggacatg
420ggcttcggtc t 43173322DNAAspergillus glaucus 73gcgttggtgc
acgacggtac caaggggact ctcatactcc ttaattctct ggccggtcca 60gtgtcgcgac
tcrtatggag cacattagca gatatcccac agagctacca tgtcgtccag
120gctctttgtc tgctttgcac atggcctttt ccaaccagca gtacctccac
agacccgacg 180ttcatgctgt gtggtatgat gatgcaagtc gccatgcaac
ttggtcttca ccggccttcg 240cacactcagg actttascaa gttcacagtg
gagctgattg aggaggagct cagggataaa 300gtgaggacat gggcttcggt ct
32274321DNAAspergillus glaucus 74gcgttggagc acgactgtac caggatactc
atcatctcct taattttctg gccggtccag 60cgycgcgact agtatggagc acattatcag
acatccgaca gagctaccat gtcgtaaagg 120ctctttgtct gctctgcaca
tggccttttc cgaccagcag tacctccaca gaccctackt 180ttatgctgtg
tggtatgatg atgcaagtcg ccatgcagct tggtcttcac cggccttcgc
240acactcagga ctttagcaag tttacagtgg agctgattga ggaggaacta
agggacaaag 300tgaggacatg ggcttcggtc t 32175349DNAAspergillus
versicolor 75cgcccttgct ggcaaccaga gcccctctgc ttccgatgtt aaggaggttc
tctcctccgt 60cggtgttgac gctgactctg agcgcctcga gaagctcatc gctgagctcc
agggcaagga 120catcaacgag gttcgttttg acagaaccgc ttgaaattct
tggcagcaga ctaatgaaac 180attttctgca gttgatcgct gagggtacca
ctaagctcgc ttccgttccc tctggcggtg 240ctggtgctgc tgctgctccc
gctgctggtg gcgctgctgc cgctgaggcc cctgccgccg 300agaaggagga
ggagaaggag gagtccgacg aggacatggg cttcggtct 34976302DNACandida
lusitaniae 76ktattgttgg kcaacgctgg taacaccgcc ccatctgctg ctgacgtcaa
gaaggtcttg 60gaatccgtct ctattgaggt tgaggacgac aaggttgaga agttgttggc
tgaagttgaa 120ggcaagaacg tcgaagagtt gattgccgag ggtaacgaga
agttgtcttc tgttccatct 180ggtgctccag ctgctgctgg tgccgctgct
gcttctggtt ctactgaggc tgctgctgaa 240gagccacaag aagaagagaa
ggaggagtct gacgacgaca tgggtttcgg tttattcgat 300ta
30277286DNACandida famata 77cttctccatc cgcttctgac atcagtagtt
tattagaaac cgttggtgct gaagctgacg 60aagctagaat cagtgcttta ttgaaggact
tagaaggtaa gcaagtcgct gacttaattg 120ctgaaggtca aaccaagttg
gcttccgttc caactggtgg tgctggtgct gctgctggtg 180gtgccgctgc
tgcttctggt gatgccggtg cagctgctgc tgaagaagaa aaggaagaag
240aaaaggaaga atccgacgat gacatgggtt tcggtttatt cgatta
28678282DNACandida famata 78cttctccatc agcctctgac gtcagtgctt
tattagaaac cgttggtgct gaagttgacc 60aaggtagagt tagtgcttta ttgaaggact
tagaaggtaa gcaagttgcc gacttaattg 120ctgaaggtca aaccaagtta
gcttctgtcc caaccggtgg tgctgcttct gctggtggtg 180ctgccgctgc
ttctggtgct gccggtgcag ctgctgttga agaagaaaag gaagaagaaa
240aggaagaatc cgatgaagat atgggtttcg gtttattcga tt
28279286DNACandida famata 79cttctccatc agcctctgac gtcagtgctt
tattagaaac cgttggtgct gaagttgacc 60aaggtagagt cagtgcttta ttgaaggact
tagaaggtaa gcaagtcgcc gacttaattg 120ctgaaggtca aaccaagtta
gcttctgtcc caactggtgg tgctgcttct gctggtggtg 180cttccgctgc
tgcttctggt gatgccggtg cagctgctgc tgaagaagaa aaggaagaag
240aaaaggaaga atccgatgaa gatatgggtt tcggtttatt cgatta
28680298DNACandida haemulonii 80tttcggttta ttcgattaac acttccccag
ctgcctctga catcaagaag gtgttggagt 60ctgtctccat cgaggttgag gacgacaagg
tcgagaagtt gttggctgag gtcgagggca 120agaacgccga ggagttgatt
gccgagggta acgagaagtt gtcttctgtc ccaactggtg 180ctccagctgg
tggtgctgcc gctgctggtg gtgctgctcc agaggctgct gctgagaagg
240aagaggaggc cgctgccgag gagtctgacg acgacatggg tttcggttta ttcgatta
29881302DNACandida haemulonii 81ttattgttgg tcaacgccgg taacacttcc
ccagctgcct ctgacatcaa gaaggtgttg 60gagtctgtct ccatcgaggt tgaggacgac
aaggtcgaga agttgttggc tgaggtcgag 120ggcaagaacg ccgaggagtt
gattgccgag ggtaacgaga agttgtcttc tgtcccaact 180ggtgctccag
ctggtggtgc tgccgctgct ggtggtgctg ctccagaggc tgctgctgag
240aaggaagagg aggccgctgc cgaggagtct gacgacgaca tgggtttcgg
tttattcgat 300ta 30282280DNACandida pulcherrima 82acacctctcc
atccgccgcc gatgtcaaga aggtcttgga gtccgtttcc atcgaggttg 60aggaggacaa
ggtcgagaag ttgctcgctg aggtcgaggg caagagcgtc gaggacttga
120tcgctgaggg taacgagaag ttgtcttctg tcccaactgg tggcccagcc
gccggtggtg 180ccgctgccgc tgctggtggt gacgccgctc ctgccgagga
ggccgctgag gaggccgccg 240aggagtctga cgacgacatg ggtttcggtt
tattcgatta 28083271DNACandida pulcherrima 83catccgccgc cgatgtcaag
raggtcttgg agtccgtttc catcgaggtt gaggaggaca 60agatcgagaa gttgctcgct
gaggtcgagg gcaagagcgt cgaggacttg atcgctgagg 120gtaacgagaa
gttgtcttct gtcccaactg gtggcccagc cgccggtggt gccgctgccg
180ctgccggtgg tgacgccgct cctgccgagg aggccgctga ggaggccgcc
gaggagtctg 240acgacgacat gggtttcggt ttattcgatt a 27184272DNACandida
utilis 84accgccgaca agatcacctc cgtcttggag tctgtcggta ttgaggttga
ggagtcccaa 60gtcaccgagt tgatctctgc ccttgagggt aagtccgttg aggagctcat
tgctgaaggt 120aacgagaagt tggcttctgt tccaaccggt ggtgctggtg
ctgctccagc tgccggtgcc 180ggtgctgctg atgctgatgc tccagctgag
gctgctgagg
aggctgctga ggaggagtct 240gacgatgaca tgggtttcgg tttattcgat ta
2728511DNAArtificial SequencePrimer 85gaggagcgcc t
118612DNAArtificial SequencePrimer 86gaggagcgcc tc
128713DNAArtificial SequencePrimer 87ggaggagcgc ctc
138813DNAArtificial SequencePrimer 88tgaggagcgc ctc
138914DNAArtificial SequencePrimer 89ccggagggaa cgga
149013DNAArtificial SequencePrimer 90ccggagggaa cgg
139118DNAArtificial SequencePrimer 91actacagctc gaagatta
189218DNAArtificial SequencePrimer 92acgttgaatg attgagac
189319DNAArtificial SequencePrimer 93ttgcgattac aagatggaa
199419DNAArtificial SequencePrimer 94cttcggacta ctgcgatga
199519DNAArtificial SequencePrimer 95acctctccat cagcttctg
199622DNAArtificial SequencePrimer 96acctctgtct tatcatctgt cg
229722DNAArtificial SequencePrimer 97aagaaggtta tcgaatctgt tg
229822DNAArtificial SequencePrimer 98tccgctttat tggaacaagt tg
229922DNAArtificial SequencePrimer 99tcctcattgt tggaatccgt tg
2210018DNAArtificial SequencePrimer 100tcagcaatca attcttgc
1810118DNAArtificial SequencePrimer 101ctcttcgacg gacttacc
1810218DNAArtificial SequencePrimer 102ttcgtccaag gacttacc
1810318DNAArtificial SequencePrimer 103ttcttgcaag tctttacc
1810418DNAArtificial SequencePrimer 104ctcgttgatg tctttacc
1810522DNAArtificial SequencePrimer 105accgctttat tggaatccgt tg
2210625DNAArtificial SequencePrimer 106atccgacaag ttagacaagt taatc
2510723DNAArtificial SequencePrimer 107agaatcaacg aattgttgtc tgc
2310826DNAArtificial SequencePrimer 108atcttccaaa ttagacttat tgttga
2610927DNAArtificial SequencePrimer 109gaagaatcaa gattatctac
cttgttg 27110380DNAAspergillus fumigatus 110ttacctcctc ctcgcccttg
ctggcaacac ctccccgtcc tctgaggatg tcaaggccgt 60cctctcttcc gttggcattg
atgccgatga ggagcgcctg aacaagctca ttgctgagct 120cgagggcaag
gacctccagg aggttagtaa ctacagctcg aagattacag actgggaatt
180ttggactggc gctgacatcg aactctacaa cagctcattg ccgagggttc
caccaagctc 240gcttccgttc cctccggtgg tgctgccgcc gctgctcctg
ccgctgccgg tgccgctgcc 300ggtggtgctg ctgctcctgc cgctgaggag
aagaaggagg aggagaagga ggagtccgac 360gaggacatgg gcttcggtct 380
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